Methods for treating hematological cancers and use of companion biomarkers for 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione

Abstract

Provided is a method for identifying a subject with a blood cancer who is likely to respond to a therapeutic compound, comprising administering a therapeutic compound to the subject with a blood cancer; obtaining a sample from the subject; measuring the level of a biomarker in the sample from the subject; and diagnosing the subject as being likely to respond to the therapeutic compound if the level of the biomarker in the subject's sample is altered compared to a reference level of the biomarker; wherein the therapeutic compound is Compound 1, Compound 2, or Compound 3.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 924,044, filed October 21, 2019, the entire contents of which are incorporated herein by reference.

[0002] 1. Field of the Invention Provided herein are methods for identifying and diagnosing patients with hematological cancers, such as diffuse large B-cell lymphoma (DLBCL) or chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL). In particular, provided herein are methods for measuring the expression levels of certain biomarkers to identify patients with hematological cancers, such as DLBCL or CLL / SLL, who may be responsive to treatment with a compound of formula (I) or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. Also provided herein are methods for treating such patients with a compound of formula (I) or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, incorporating the above methods. Additionally, provided herein are kits for carrying out the methods described herein. [Background technology]

[0003] 2. Background of the Invention Cancer is primarily characterized by an increase in the number of abnormal cells originating from a given normal tissue, the invasion of adjacent tissues by these abnormal cells, or the spread of malignant cells via lymph or blood to regional lymph nodes, and metastasis. Clinical data and molecular biological studies indicate that cancer growth is a multistep process that begins with minor prebiotic changes that may develop into neoplasms under certain conditions. The neoplastic lesions may evolve clonally and develop increasing capacities for invasion, proliferation, metastasis, and heterogeneity, especially under conditions that allow the neoplastic cells to escape the host's immune surveillance. Current cancer therapies include surgery, chemotherapy, hormonal therapy, and / or radiation therapy to eradicate the neoplastic cells in the patient. Recent advances in cancer therapy are reviewed by Rajkumar et al. in Nature Reviews Clinical Oncology 11, 628-630 (2014).

[0004] NHL is the fifth most common cancer in both men and women in the United States. In 2012, an estimated 385,700 patients worldwide were diagnosed with NHL, and approximately 199,700 patients died as a result of the disease (Torre, LA et al., Global cancer statistics, 2012; CA Cancer J. Clin. 65, 87-108 (2015)). Diffuse large B-cell lymphoma (DLBCL) accounts for approximately one-third of non-Hodgkin lymphomas (NHLs) and is the most common form of B-cell NHL, with an estimated 27,650 new cases of DLBCL diagnosed in the United States in 2016, accounting for approximately 26% of all diagnosed mature B-cell NHL neoplasms (Teras, LR et al., 2016 US lymphoid malignancy statistics by World Health Organization subtypes; CA Cancer J. Clin. 66, 443-459 (2016)). While some DLBCL patients are cured with traditional chemotherapy, the rest die from the disease.

[0005] The greatest obstacle to treating DLBCL with current therapies is the ability of certain lymphomas to acquire resistance to or be refractory to the standard frontline treatment, R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), or newer agents such as venetoclax and ibrutinib. Approximately 30-40% of patients develop relapsed / refractory disease, which remains the leading cause of high morbidity and mortality due to limited treatment options (Camicia et al., Mol. Cancer 14, 207(2015)). As a result, patients with relapsed / refractory DLBCL have a poor prognosis.

[0006] Recent advances in gene expression profiling have led to the identification of at least three distinct molecular subtypes of DLBCL: germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. The ABC-DLBCL subtype is associated with a very poor prognosis when treated with CHOP alone, and the majority of ABC-DLBCL patients treated with CHOP alone will succumb to their disease. The 3-year progression-free survival (PFS) and overall survival (OS) rates of patients with ABC-DLBCL treated with R-CHOP were approximately 40% and 45%, respectively, whereas the corresponding PFS and OS rates of patients with GCB-DLBCL treated with R-CHOP were approximately 74% and 80%, respectively. There is a particularly urgent clinical need in these patient groups due to their highly aggressive clinical course, high chemorefractory nature, and poor overall survival when treated with R-CHOP.

[0007] In addition, disease progression in lymphoma patients is associated with a decline in immune system function. For example, T cell exhaustion has been observed in patients with B-cell non-Hodgkin's lymphoma (NHL) (Yang, 2014; Yang, 2015). T cell exhaustion indicates a decline in differentiation, proliferation, and the ability to produce cytokines. Therefore, improving immune system activation may be useful in the treatment of hematological cancers such as DLBCL.

[0008] Cytotoxic chemotherapy predictably suppresses the hematopoietic system and impairs host defense mechanisms, and the toxicity associated with cancer chemotherapy is severe. The degree and duration of neutropenia determine the risk of infection (Crawford, 2004). As a result, preclinical assessment of myelotoxicity remains important for developing new therapeutic options for patients with hematological cancers. Neutrophils, as the first cellular component of the inflammatory response and a key component of innate immunity, represent the first line of defense against infection. Moreover, neutropenia blunts the inflammatory response to early infections, allowing bacteria to proliferate and infiltrate. Thus, assessment of neutropenia can help monitor the treatment of hematological cancers, such as treatment strategies in DLBCL or CLL / SLL.

[0009] Chronic lymphocytic leukemia (CLL) is a lymphoproliferative malignancy characterized by the progressive accumulation of morphologically mature but functionally insufficient B lymphocytes with distinct clusters of differentiation (CDs) CD19+, CD5+, and CD23+ phenotypes in blood, bone marrow, and lymphoid tissues. It is the most common leukemia in North America and Europe with an incidence rate of 4.0 per 100,000 per year, affecting mostly older patients with a median age at onset of 72 years. The clinical course of CLL ranges from an indolent disease with prolonged survival of more than 12 years to an aggressive disease with a median survival of 2 years, influenced by the stage at presentation and certain disease-specific features such as cytogenetic abnormalities. The current clinical course and prognosis reflect the evolution of the therapeutic landscape, including the emergence of new agents available for the treatment of CLL. Despite the recent introduction of several highly effective agents, CLL remains an incurable disease for patients who do not undergo allogeneic stem cell transplantation, thus necessitating the development of alternative and additional therapeutic options.

[0010] The molecular pathogenesis of CLL / SLL represents a complex and multifaceted process characterized by specific genetic abnormalities, alterations in cell signaling pathways, including B cell receptors and apoptotic pathways, and the convergence of the tumor-immune microenvironment. The term CLL is used when the disease is primarily manifested in the blood, whereas the term small lymphocytic lymphoma (SLL) is used when the disease is primarily metastatic to lymph nodes. Specifically, SLL, as defined by the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) criteria, is a disease in patients who are otherwise diagnosed with CLL, but who have relatively normal peripheral lymphocyte counts and require the presence of lymphadenopathy and / or splenomegaly. In contrast to CLL, which is more prevalent in blood and bone marrow, as well as other disease sites such as lymph nodes, spleen, and extranodal sites, patients with SLL have less prominent signs in the peripheral blood.

[0011] The CLL treatment landscape is evolving, as evidenced by the recent approval of several novel targeted agents, such as ibrutinib and venetoclax. However, despite the availability of these new agents, patients continue to relapse or are refractory to treatment. Moreover, patients with low-risk cytogenetic features continue to show worse outcomes compared to patients without these features. Improved and novel combination therapies for CLL will continue to be a significant medical need. In addition, the increased use of targeted therapeutics has led to the emergence of new mutations that confer resistance to treatment. For example, resistance to the BTK inhibitor ibrutinib is associated with either mutations at the BTK binding site or mutations that result in autonomous B-cell receptor activity. Therefore, exploring agents with novel mechanisms is important to provide treatment options with unique mechanisms of action (MOA) for patients who develop resistance to the emergence of targeted agents.

[0012] There remains a significant need for safe and effective methods of treating, preventing and managing hematological cancers such as DLBCL or CLL / SLL, while reducing or avoiding the toxicity and / or side effects associated with conventional therapeutic agents, particularly for DLBCL or CLL / SLL that are refractory to standard therapies. The present invention meets this need and provides related advantages as well.

[0013] Citation or identification of any reference in this section of this application shall not be construed as an admission that such reference is prior art to the present application. Summary of the Invention

[0014] 3. Summary of the Invention In one aspect, provided herein is a method for identifying a subject with a hematological cancer that is likely to be responsive to a therapeutic compound, or predicting the responsiveness of a subject with or suspected of having a hematological cancer to a therapeutic compound, comprising: (a) obtaining a sample from said subject; (b) measuring the level of a biomarker in the sample; (c) Suppose, (i) the level of the biomarker in the sample is detectable; or (ii) if the level of the biomarker in the sample is altered compared to the level of the reference biomarker; diagnosing the subject as likely to be responsive to the therapeutic compound; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0015] In another aspect, provided herein is a method of selectively treating hematological cancer in a subject with hematological cancer, comprising: (a) obtaining a sample from a subject with blood cancer; (b) measuring the level of a biomarker in the sample; (c) Suppose, (i) the level of the biomarker in the sample is detectable; or (ii) if the level of the biomarker is altered compared to a reference level of the biomarker; diagnosing the subject as likely to be responsive to the therapeutic compound; and (d) administering a therapeutically effective amount of a therapeutic compound to a subject diagnosed as likely to be responsive to said therapeutic compound; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0016] In some embodiments of the methods provided herein, the biomarker is cereblon (CRBN), and the methods include diagnosing the subject as likely to be responsive to the therapeutic compound if CRBN is detectable or higher than a control level in the sample.

[0017] In other embodiments of the methods provided herein, the biomarker is Ikaros, Aiolos, ZFP91, or a combination thereof, and the method includes diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is lower than the control level. In some embodiments, the biomarker is a combination of Ikaros and Aiolos, and the method includes diagnosing the subject as likely to be responsive to the therapeutic compound if the levels of both Ikaros and Aiolos are lower than their respective control levels.

[0018] In another aspect, provided herein is a method for identifying a subject with a hematological cancer that is likely to be responsive to a therapeutic compound, or predicting the responsiveness of a subject with or suspected of having a hematological cancer to a therapeutic compound, comprising: (a) obtaining a sample from said subject; (b) adding said therapeutic compound to said sample; (c) measuring the level of a biomarker in the sample; and (d) diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is altered compared to the level of a reference biomarker; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0019] In yet another aspect, provided herein is a method for identifying a subject with hematological cancer who is likely to be responsive to a therapeutic compound, or predicting the responsiveness of a subject with or suspected of having hematological cancer to a therapeutic compound, comprising: (a) administering to a subject a therapeutic compound; (b) obtaining a sample from the subject; (c) measuring the level of a biomarker in the sample; and (d) diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is altered compared to the level of a reference biomarker; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0020] In another aspect, provided herein is a method for monitoring the efficacy of a therapeutic compound in treating a hematological cancer in a subject, comprising: (a) administering a therapeutic compound to a subject; (b) obtaining a sample from the subject; (c) measuring the level of a biomarker in the sample; and (d) comparing the level of the biomarker in the sample to a reference level of the biomarker, wherein an alteration in the level of the biomarker indicates efficacy of the therapeutic compound in treating the hematological cancer in the subject; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0021] In another aspect, provided herein is a method for adjusting the dosage or frequency of treatment of a subject with a hematological cancer with a therapeutic compound, comprising: (a) administering to a subject a dose of a therapeutic compound; (b) obtaining one or more samples from the subject at different time points; (c) monitoring the level of the biomarker in one or more samples; and (d) adjusting a dosage for subsequent administration of the therapeutic compound to the subject based on the altered level of the biomarker in the reference sample; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0022] In some embodiments, the methods provided herein further comprise administering a therapeutically effective amount of a therapeutic compound to a subject diagnosed as likely to be responsive to the therapeutic compound.

[0023] In certain embodiments, the altered level of the biomarker in the sample is higher than the reference level of the biomarker, hi other embodiments, the altered level of the biomarker in the sample is lower than the reference level of the biomarker.

[0024] In some embodiments, an increase in the level of the biomarker relative to the level of the reference biomarker indicates the efficacy of the therapeutic compound in treating a hematological cancer in the subject. In other embodiments, a decrease in the level of the biomarker relative to the level of the reference biomarker indicates the efficacy of the therapeutic compound in treating a hematological cancer in the subject.

[0025] In any one of the embodiments of the methods provided herein, the reference biomarker level is the level of the biomarker in the reference sample obtained from the subject before administering the therapeutic compound to the subject, and the reference sample is from the same source as the sample.In other embodiments, the reference biomarker level is the level of the biomarker in the reference sample obtained from a healthy subject without blood cancer, and the reference sample is from the same source as the sample.In certain embodiments, the reference biomarker level is the level of a predetermined biomarker.

[0026] In certain embodiments of the methods provided herein, the biomarker comprises a marker of apoptosis, and alteration of the level of the biomarker is indicative of induction of apoptosis. In a specific embodiment, the biomarker is selected from cleaved caspase 3, cleaved caspase 7, cleaved poly(ADP-ribose) polymerase (PARP), BCL2, survivin, phosphatidylserine (PS) and DNA, Bcl-2-like protein 11 (BIM), tumor necrosis factor (TNF), interleukin-10 (IL-10), or interleukin-27 (IL27), or a combination thereof. In certain embodiments, the biomarker comprising a marker of apoptosis is selected from the group consisting of annexin-V, 7-amino-actinomycin D (7-AAD), and deep red anthraquinone 7 (DRAQ7), or a combination thereof. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0027] In some embodiments of the methods provided herein, the biomarker is selected from IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, sIL-2, sIL-6, collagen-I and -III, PAI-1, CD69, or sIL-10, or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0028] In certain embodiments of the methods provided herein, the biomarker is associated with interferon signaling. In specific embodiments, the biomarker associated with interferon signaling includes interleukin-6 signal transducer (IL6ST), interferon-induced transmembrane protein 3 (IFITM3), interferon alpha-inducible protein 6 (IFI6), 2'-5'-oligoadenylate synthetase 3 (OAS3), interferon alpha (IFNα), interferon beta (IFNβ), or a combination thereof. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0029] In some embodiments, the biomarker is associated with cytokine / chemokine signaling.In a specific embodiment, the biomarker associated with cytokine / chemokine signaling comprises interleukin-23 subunit alpha (IL23A), CC motif chemokine 1 (CCL1), or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0030] In certain embodiments, the biomarker is associated with cell adhesion.In particular embodiments, the biomarker associated with cell adhesion comprises E-selectin (SELE), P-selectin glycoprotein ligand 1 (SELPLG), thromboxane A2 (TXA2), or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0031] In some embodiments, the biomarker is associated with cell-cell junction.In a specific embodiment, the biomarker associated with cell-cell junction includes claudin 7 (CLDN7), claudin 12 (CLDN12), or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0032] In certain embodiments, biomarker is G-protein coupled receptor.In particular embodiments, G-protein coupled receptor includes free fatty acid receptor 2 (FFAR2).In some embodiments, the biomarker in sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in sample is lower than the reference level of the biomarker.

[0033] In some embodiments of the method provided herein, biomarker is associated with extracellular matrix.In a specific embodiment, the biomarker associated with extracellular matrix comprises CD209, SERPINA, SERPINB7, or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0034] In certain embodiments, the biomarker is associated with cell cycle. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0035] In some embodiments, the biomarker is associated with transcription. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0036] In some embodiments of the methods provided herein, the biomarkers are Aiolos (IKZF3), Ikaros (IKZF1), E3 ubiquitin-protein ligase ZFP91 (ZFP91), protein C-ets-1 (ETS1), Max binding protein. MNT (MNT), myocyte-specific enhancer factor 2B (MEF2B), snRNA activator protein complex subunit 1 (SNAPC1), lysine-specific demethylase 4B (KDM4B), transcription factor AP-4 (TFAP4), nuclear pole transcription factor 1 (UBTF), bromo-adjacent homology domain-containing 1 protein (BAHD1), methyl-CpG-binding domain protein 4 (MBD4), chromobox protein homolog 2 (CBX2), tumor protein 63 (TP63), transducin-like enhancer protein 3 (TLE3), forkhead box protein P1 (FOXP1), zinc finger and BTB domain-containing protein 11 (ZBTB11), interferon regulatory factor 4 (IRF4), mediator of RNA polymerase II transcription subunit 26 (MED26), cyclic AMP-dependent transcription factor ATF-7 (ATF7) , zinc finger protein 644 (ZNF644), lysine-specific demethylase 5B (KDM5B), upstream stimulatory factor 2 (USF2), transcription factor 25 (TCF25), lysine-specific demethylase 4A (KDM4A), lethal (3) malignant brain tumor-like protein 2 (L3MBTL2), nRNA-activator protein complex subunit 4 (SNAPC4), lysine-specific demethylase 5 (KDM5), transcription factor COE1 (EBF1 ), forkhead box protein J2 (FOXJ2), nuclear factor of activated T cells cytoplasm 1 (NFATC1), activator of mRNA downregulation protein ZFP36 (ZFP36), hepatocellular carcinoma-inducible growth factor (HDGF), ETS-related transcription factor Elf-1 (ELF1), promyelocytic leukemia protein (PML), Myb-related protein BMYBL2, and mothers against decapentaplegic homolog 2 (M2P2).2) (SMAD2), chromodomain-helicase-DNA-binding protein 2 (CHD2), signal transducer and activator of transcription 1 (STAT1), paired box protein Pax5 (PAX5), signal transducer and activator of transcription 2 (STAT2), pygopath homolog 2 (PYGO2), interferon regulatory factor 9 (IRF9), polycomb group RING finger protein 2 (PCGF2), and cyclic AMP-dependent transcription factor ATF-3 (ATF3). In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0037] In some embodiments of the methods provided herein, the biomarker comprises one or more genes selected from the group consisting of interleukin-23 subunit alpha (IL23A), CC motif chemokine 2 (CCL2), and SLIT-ROBO Rho GTPase-activating protein 1 (SRGAP1).In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0038] In certain embodiments, the biomarker comprises a CRBN binding protein or a transcriptional target protein of the CRBN binding protein. In a specific embodiment, the CRBN binding protein comprises IKAROS, AIOLOS, or ZFP91. In other specific embodiments, the transcriptional target protein of the CRBN binding protein comprises BCL6, c-MYC, or IRF4. In further embodiments, the transcriptional target protein of the CRBN binding protein comprises an interferon-inducible gene. In a specific embodiment, the interferon-inducible gene comprises interferon regulatory 7 (IRF7), interferon-induced protein with tetratricopeptide repeat unit 3 (IFIT3), DEAD box protein 58 (DDX58), or a combination thereof. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0039] In some embodiments of the method provided herein, the biomarker is selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21).In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0040] In certain embodiments of the methods provided herein, the biomarker comprises a T cell activation marker.In a specific embodiment, the T cell activation marker comprises a T cell activation-associated cytokine.In some embodiments, the T cell activation-associated cytokine comprises interleukin 2 (IL-2).In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0041] In some embodiments, the biomarker comprises PD1 and LAG3. In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0042] In some embodiments of the method provided herein, the biomarker comprises effector cytokine or effector chemokine.In a specific embodiment, the effector cytokine or effector chemokine comprises granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), or a combination thereof.In some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker.

[0043] In certain embodiments, the biomarker is expressed in white blood cells. In a specific embodiment, the white blood cells include lymphoid cells. In a more specific embodiment, the lymphoid cells include T cells.

[0044] In another aspect, provided herein is a method of treating hematological cancer, comprising: (a) obtaining a first sample from a subject with blood cancer; (b) measuring the level of a biomarker in the first sample; (c) administering to the subject a therapeutically effective amount of a therapeutic compound; (d) obtaining at least one additional sample from the subject after administration of the treatment; and (e) measuring the level of the biomarker in at least one additional sample; if the level of the biomarker in the at least one additional sample is at or near the level of the biomarker in the first sample, then administering to the subject another therapeutically effective amount of the therapeutic compound; wherein the therapeutic compound has formula (I): [ka] or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0045] In certain embodiments of the method of treating hematological cancer, the biomarker comprises Ikaros. In a specific embodiment, the Ikaros biomarker is expressed in white blood cells. In a more specific embodiment, the white blood cells include myeloid cells. In some embodiments, the myeloid cells include neutrophils. In a specific embodiment, the biomarker comprises CD11b + , CD34 - , and CD33 - The phenotype of the neutrophils is

[0046] In any embodiment of any one of the methods provided herein, the compound of formula (I) includes (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a tautomer, isotopologue, or a pharmaceutically acceptable salt thereof. In other embodiments of any of the methods provided herein, the compound of formula (I) includes (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a tautomer, isotopologue, or a pharmaceutically acceptable salt thereof. In some embodiments of any of the methods provided herein, the compound of Formula (I) includes a mixture of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione and (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0047] In further embodiments of any of the methods provided herein, the method comprises administering a therapeutically effective amount of a second active agent or supportive care treatment. In certain embodiments, the second active agent includes an HDAC inhibitor (e.g., panobinostat, romidepsin, vorinostat, or citalinostat), a BCL2 inhibitor (e.g., venetoclax), a BTK inhibitor (e.g., ibrutinib or acalabrutinib), an mTOR inhibitor (e.g., everolimus), a PI3K inhibitor (e.g., idelalisib), a PKCβ inhibitor (e.g., enzastaurin), a SYK inhibitor (e.g., fostamatinib), a JAK2 inhibitor (e.g., fedratinib, pacritinib, ruxolitinib, baricitinib, gandotinib, lestaurtinib, or momelotinib), an Aurora A kinase inhibitor (e.g., alisertib), an EZH2 inhibitor (e.g., tazemetostat, GSK126, CPI-12, 05, 3-deazaneplanocin A, EPZ005687, EI1, UNC1999, or sinefungin), BET inhibitors (e.g., bilabresib or 4-[2-(cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one), hypomethylating agents (e.g., 5-azacytidine or decitabine), chemotherapeutic agents (e.g., bendamustine, doxorubicin, etoposide, methotrexate, cytarabine, vincristine, ifosfamide, melphalan, oxaliplatin, or dexamethasone), anti-CD20 monoclonal antibodies (e.g., rituximab, or obinutuzumab), or epigenetic compounds, or combinations thereof. In some embodiments, the second active agent comprises rituximab. In certain embodiments, the second active agent comprises obinutuzumab.

[0048] In certain embodiments of any one of the methods provided herein, the hematological cancer affects hematopoietic tissue or lymphoid tissue. In some embodiments, the hematological cancer includes non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma includes diffuse large B-cell lymphoma (DLBCL). In a more specific embodiment, the DLBCL is relapsed, refractory, or resistant to conventional therapy. In other embodiments, the hematological cancer includes chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In a specific embodiment, the CLL / SLL is relapsed or refractory CLL / SLL.

[0049] In certain embodiments of any one of the methods provided herein, the sample contains hematological cancer cells.

[0050] In any embodiment of any one of the methods provided herein, measuring the level of the biomarker includes measuring the protein level of the biomarker.

[0051] In any embodiment of any one of the methods provided herein, measuring the level of the biomarker includes measuring the mRNA level of the biomarker.

[0052] In certain embodiments of any one of the methods provided herein, measuring the level of the biomarker includes measuring the cDNA level of the biomarker. [Brief description of the drawings]

[0053] 4. Brief description of the drawings [Figure 1]Figure 1A-1C show that DLBCL cell lines expressed DLBCL-associated proteins Myc, BCL2, and BCL6 (Figure 1A); as well as CRL4CRBN E3 ubiquitin ligase and its substrates Aiolos, Ikaros, and ZFP91 (Figure 1B). Quantification of CRBN expression levels was normalized to CRBN levels in DF15 cells (Figure 1C). Beta-tubulin was used as a loading control.

[0054] [Diagram 2] Compound 1 was active in DLBCL cell lines that had acquired resistance to doxorubicin. Viability and apoptosis induction curves for the parental cell lines (squares) of ABC (top panel) and GCB (bottom panel) cell lines and their related doxorubicin-R cell lines (circles) were generated following exposure to serial dilutions of Compound 1 for 5 days.

[0055] [Diagram 3] Representative immunoblots showing the expression profiles of cereblon, and related cereblon substrates Aiolos, Ikaros, and ZFP91, and c-Myc, IRF4, BCL2, MCL1, and BCL6 proteins in cell lines that have acquired doxorubicin resistance and their corresponding matched parental cells are shown.

[0056] [Figure 4]Figures 4A and 4B show that Compound 1 was selectively anti-proliferative in endothelial cells, T- and B-lymphocytes (Figure 4A), and in coronary smooth muscle cells and fibroblasts (Figure 4B). The BioMAP Diversity PLUS Panel was evaluated after treatment with Compound 1. The X-axis lists the quantitative protein-based biomarker readouts measured in each system. The Y-axis shows the log-transformed ratio of biomarker readouts in drug-treated samples (n=1) relative to vehicle control (n>6). The grey area around the Y-axis indicates the 95% significance limits generated from the vehicle control used so far. Biomarker activity was annotated when two or more consecutive concentrations changed in the same direction relative to vehicle control, were outside the significance limits, and at least one concentration had an effect magnitude of >20% (log10 ratio >0.1). Anti-proliferative effects are indicated by thick grey arrows.

[0057] [Diagram 5]Figures 5A-5D show that the activity of Compound 1 is dependent on CRBN expression. (Figure 5A) shows that the protein expression of Aiolos, Ikaros, ZFP91, IRF4, and c-Myc was decreased and apoptotic proteins (cleaved caspase 7, cleaved caspase 3, and cleaved PARP), as well as the interferon-stimulated gene IFIT3, were induced in SU-DHL2 cells expressing CRBN in a time- and concentration-dependent manner; (Figure 5B) shows that the decreased levels of Aiolos, Ikaros, and ZFP91, as well as the increased levels of cleaved caspase 3, cleaved caspase 7, and cleaved poly(ADP-ribose) polymerase (PARP) were quantified, first normalized to β-tubulin and then further normalized to the levels of each protein at 0 hours after treatment with DMSO (circles), 0.001 nM (squares), 0.01 nM (upward triangles), or 0.1 nM (downward triangles) of Compound 1; (Figure 5C) shows that the protein expression of Aiolos, Ikaros, and ZFP91 was decreased and apoptotic proteins (cleaved caspase 7, cleaved caspase 3, and cleaved caspase 7), as well as the interferon-stimulated gene IFIT3, were induced in SU-DHL2 cells expressing CRBN in a time- and concentration-dependent manner; No effect was observed in CRBN knockout cells; (FIG. 5D) shows that apoptosis was increased over time using fluorescent caspase-3 reagent in SU-DHL2CRBNWT cells (top panel) but not in SU-DHL2CRBN− / − cells (bottom panel) following treatment with 0.01 nM (circles), 0.1 nM (squares), 1.0 nM (upper triangles), 10 nM (downper triangles), 100 nM (diamonds), or 1000 nM (open circles) of compound 1. WT = wild type

[0058] [Figure 6]Figures 6A-6D show that Compound 1 reduced the expression of CRBN substrate proteins and induced the expression of apoptosis and interferon-stimulated genes in DLBCL cell lines. (Figures 6A and 6C) show that the protein expression of Aiolos, Ikaros, ZFP91, IRF4, and c-Myc was reduced, and apoptosis proteins (cleaved caspase 7, cleaved caspase 3, and cleaved PARP), as well as interferon-stimulated genes, IRF7, DDX58, and IFIT3, were induced in TMD8 and Karpas-422 cells expressing CRBN, respectively, in a time- and concentration-dependent manner. (FIG. 6B) shows that after 24 hours of treatment with Compound 1, decreased expression of Aiolos, Ikaros, ZFP91, BCL6, IRF4, c-Myc, anti-apoptotic protein BIM, and survivin, and induction of pro-apoptotic protein (cleaved caspase 7), as well as interferon-stimulated genes IRF7, DDX58, and IFIT3, were observed in TMD8 cells; (FIG. 6D) shows that after 24 hours of treatment with Compound 1, decreased expression of Aiolos, Ikaros, ZFP91, and BCL6 (left panel), followed by induction of pro-apoptotic proteins cleaved caspase 3, cleaved caspase 7, and cleaved PARP at 48 hours, as well as induction of p21, IRF7, IFIT3, DDX58 (middle panel), and decreased anti-apoptotic proteins survivin and BCL2, as well as MYC at 72 hours (right panel).

[0059] [Figure 7]Figures 7A and 7B show that the cellular fitness of DLBCL cell lines was dependent on individual cereblon substrates. (Figure 7A) is a schematic diagram showing the design of a flow cytometry-based cellular competition assay to evaluate the relative change in cellular fitness upon knockout of a gene of interest. (Figure 7B) shows the RFP+ / GFP+ ratios in KARPAS-422-Cas9, U-2932-Cas9, RIVA-Cas9, SU-DHL-16-Cas9, HT-Cas9, and SU-DHL-4-Cas9 cell lines for sgNT-1 (circles and solid line), sgNT-2 (circles and dashed line), sgNC-1 (diamonds and solid line), sg Relative cellular fitness is shown for sgIKZF1-1 (upward triangle and solid line), sgIKZF1-2 (upward triangle and dashed line), sgIKZF3-1 (square and solid line), sgIKZF3-2 (square and dashed line), sgZFP91-1 (downward triangle and solid line), sgZFP91-3 (downward triangle and dashed line), and sgETF1-1 ("X" and solid line). Cells were normalized to the percentage of RFP+ / GFP+ in sgNT-1 cells at each time point. Knockout of ETF1 (sgETF1-1) serves as a positive control for a robust reduction in the percentage of RFP+ / GFP+. Error bars indicate the standard error of the mean of three independent experiments.

[0060] [Figure 8] Figures 8A and 8B show that loss of Ikaros, Aiolos, and ZFP91 sensitizes DLBCL cells to compound 1 as measured by a flow cytometry-based cell competition assay that assesses the relative change in cell fitness upon knockout of a gene of interest. (Figure 8A) KARPAS-422-Cas9, and (Figure 8B) SU-DHL-4-Cas9 cells. DMSO = dimethylsulfoxide; GFP = green fluorescent protein; RFP = red fluorescent protein; gene knockouts are indicated above each set of RFP+ / GFP+ ratios. Error bars indicate standard error of the mean of three independent experiments.

[0061] [Figure 9]Figure 1 shows that treatment of DLBCL cells with compound 1 was equivalent to genetic knockout of the CRBN substrates Ikaros, Aiolos, and ZFP91 as measured by immunoblot. KARPAS-422-Cas9 (top left), U-2932-Cas9 (top right), and SU-DHL-4-Cas9 (bottom left); DMSO = dimethyl sulfoxide.

[0062] [Figure 10] Figure 10A and Figure 10B show that double knockout of Ikaros and Aiolos had a greater inhibition of cell fitness in DLBCL cell lines than single knockout of Ikaros and Aiolos. Figure 10A shows a schematic diagram of designing a flow cytometry-based cell competition assay to evaluate the relative change in cell fitness upon knockout of a gene of interest. Figure 10B shows the relative change in cell fitness of sgNT-1+sgNT-2 (black circle), sgIKZF1- normalized to the percentage of RFP+ / GFP+ on the first day in KARPAS-422-Cas9, U-2932-Cas9, RIVA-Cas9, SU-DHL-16-Cas9, HT-Cas9, and SU-DHL-4-Cas9 cell lines. Relative cellular fitness of sgIKZF1+sgNT-1 (upward triangle and solid line), sgIKZF1-1+sgNT-2 (upward triangle and dashed line), sgIKZF3-1+sgNT-1 (filled square and solid line), sgIKZF3-1+sgNT-2 (filled square and dashed line), sgIKZF1-1+sgIKZF3-1 (open circle with solid line), and sgIKZF1-2+sgIKZF3-2 (open circle with dashed line) cells is shown. Error bars indicate standard error of the mean of three independent experiments.

[0063] [Figure 11] Figure 1 shows that ectopic expression of degradation-resistant mutants of Ikaros (IKZF1-G151A) (up triangles), Aiolos (IKZF3-G152A) (squares), and ZFP91 (ZFP91-G405A) (down triangles) provides protection from compound 1 in KARPAS-422, RIVA, HT, and SU-DHL-4 cell lines, as measured by luciferase.

[0064] [Figure 12] Figure 1 shows that compound 1 induced degradation of Ikaros in peripheral blood mononuclear cells (PBMCs) from four healthy donors (HD1-4). After continuous exposure to compound 1 for 3, 4 or 7 days in four donors, the percentage of Ikaros positive cells was normalized to DMSO control. Ikaros was measured by flow cytometry. Data represent the mean percentage of cells positive for Ikaros. Error bars represent standard error of the mean (SEM). N is four donors in triplicates.

[0065] [Figure 13] Figure 1 shows that compound 1 increased absolute levels of interleukin-2 (IL2) secretion after exposure of healthy donor PBMCs to compound 1 for 3 days (circles), 4 days (squares) and 7 days (diamonds). Supernatants were diluted 1:10 and IL-2 secretion was measured by the MSD method. Data points represent the mean of n=3 triplicates. Error bars represent standard error of the mean (SEM). DMSO=dimethylsulfoxide; HD=healthy donor; IL-2=interleukin-2; mL=milliliters; nM=nanomoles; pg=picograms.

[0066] [Figure 14] Figure 1 shows that compound 1 increased the fold change in interleukin-2 (IL2) secretion compared to DMSO after exposure of healthy donor PBMCs to compound 1 for 3 days (circles), 4 days (squares) and 7 days (diamonds). Supernatants were diluted 1:10 and IL-2 secretion was measured by the MSD method. Data points represent the mean of n=3 triplicates. Error bars represent standard error of the mean (SEM). DMSO=dimethylsulfoxide; HD=healthy donor; IL-2=interleukin-2; mL=milliliters; nM=nanomoles; pg=picograms.

[0067] [Figure 15]Figures 15A-D show that compound 1 restimulated T cells in a Staphylococcal Enterotoxin B exhaustion assay. Figures 15A and 15C show a schematic of the SEB-induced T cell exhaustion assay. Briefly, PBMCs were treated with 100 ng / mL SEB for 72 hours and the T cell exhaustion phenotype was assessed for PD-1 and LAG3 expression by FACS. Figures 15B and 15D show the expression levels of PD-1 and LAG3 in control and SEB-treated cells. FACS = fluorescence activated cell sorting; PBMC = peripheral blood mononuclear cells; SEB = Staphylococcal Enterotoxin B; sups = supernatant.

[0068] [Figure 16] Figure 2 shows that compounds 2 and 3 reduced the expression of Ikaros, Aiolos, and ZFP91 in SU-DHL-2 cells 1, 2, or 6 hours after treatment with vehicle control (0.1% DMSO), compound 1 (1, 10, 100 nM), as measured by immunoblot.

[0069] [Figure 17] Figure 2 shows that after exposure for 45 minutes, 60 minutes, 90 minutes, or 3 hours, compound 1 (circles and solid line) and compound 2 (triangles and dashed line) degraded Ikaros in a concentration- and time-dependent manner in DF-15 cells expressing Enhanced ProLabel (ePL)-Aeolus, ePL-Ikaros, or ePL-ZFP91.

[0070] [Figure 18] Figures 18A and 18B show that compound 1 did not affect neutrophil precursor (CD34+) cell survival, as measured by annexin V and 7-actinomycin D (7-AAD), after exposure to DMSO (filled circles), 0.1 nM (squares), 1 nM (upward triangles), 10 nM (downward triangles), 100 nM (diamonds), or 1000 nM (open circles) of the compound for 14 days (Figure 18A) or 5 days starting on day 9 (Figure 18B).

[0071] [Figure 19] Figures 19A and 19B show that the percentage of mature (stage IV) cells rebounded after CD34+ cells derived from bone marrow of healthy donors were first exposed ex vivo to Compound 1 for 14 days (Figure 19A) or for 5 days starting on day 9 (Figure 19B), followed by washing and reincubation for an additional 7 days in the absence of Compound 1. Data show the percentage of stage IV cells, defined as CD34- / CD33- / CD11b+.

[0072] [Figure 20] Figures 20A and 20B show the recovery of mature (stage IV) cells after exposure of healthy donor bone marrow CD34+ cells to 0.1 nM (squares), 1 nM (upward triangles), or 10 nM (downward triangles) of Compound 1 for 14 days (Figure 20A) or 5 days starting on day 9 (Figure 20B). DMSO (circles) served as a control. Data show the percentage of stage IV healthy donor bone marrow CD34+ cells defined as CD34- / CD33- / CD11b+. The thick black line indicates 50% stage IV cells in the DMSO control.

[0073] [Figure 21] Figure 1 shows that Ikaros was first inhibited after 14 days of exposure to compound 1, then restored after washing out compound 1. After cells from donors #1, #2, and #3 were treated with consecutive exposure to compound 1 for 14 days and washed out for 1 week, the percentage of Ikaros inhibition was compared to DMSO control. Ikaros was measured by flow cytometry every 2 or 3 days. DMSO = dimethylsulfoxide; nM = nanomolar.

[0074] [Figure 22]The percentage of Ikaros inhibition is shown to be inhibited only after exposure to compound 1 for 5 consecutive days starting on day 9, followed by recovery by treatment of cells from donor 1 followed by a 1-week washout period. Ikaros was measured by flow cytometry every 2 or 3 days. DMSO = dimethylsulfoxide; nM = nanomolar.

[0075] [Diagram 23] Figure 1 shows that Ikaros protein inhibition and recovery correlates with the percentage of stage IV population during ex vivo myeloid differentiation of CD34+ bone marrow derived cells exposed to 0.1 nM (open triangles downwards), 1.0 nM (squares), 10 nM (upward triangles), 100 nM (diamonds), or 1000 nM (closed triangles downwards) of compound 1 for 14 days and washed for 1 week after treatment. The graph shows the percentage of stage IV cells defined as CD34- / CD33- / CD11b+ (solid line) versus the percentage of Ikaros protein inhibition (dashed line) compared to DMSO (circles) control cultures treated for 14 days. Both parameters were measured by flow cytometry every 2 or 3 days. Data shown are from 3 donors.

[0076] [Figure 24] We present a causal flow network model to infer the effects of Compound 1 in a DLBCL model. Proteomic effects were measured at 6 and 18 hours, and transcriptional effects were measured at 12, 24, and 48 hours, and the results were integrated to identify pathways modulated by Compound 1 treatment.

[0077] [Diagram 25]Exemplary pathways and genes involved in response to treatment with Compound 1 are shown, such as genes associated with interferon signaling (e.g., IL6ST, IFITM3, IFI6, OAS3, interferon α / β signaling), cytokine / chemokine signaling (e.g., IL23A, CCL1), apoptosis (e.g., IL27, TNF, IL10, caspases), cell adhesion (e.g., SELE, SELPLG, TXA2PA), cell-cell junctions (e.g., CLDN7, CLDN12), G-protein coupled receptors (e.g., FFAR2), extracellular matrix (e.g., CD209, SERPINA, SERPINB7), cell cycle and transcription.

[0078] [Figure 26] Figures 26A-C show exemplary gene responses after 12, 24, or 48 hours of treatment with Compound 1. Expression of IL23A (Figure 26A), CCL2 (Figure 26B), and SRGAP1 (Figure 26C) were all increased after treatment with Compound 1 in sensitive cell lines compared to intermediate or resistant cell lines.

[0079] [Figure 27] Protein expression levels of a panel of differentially expressed genes after treatment with Compound 1 for 6 or 18 hours are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] 5. Detailed Description of the Invention The methods provided herein are based, in part, on the discovery that changes in, e.g., increased and / or decreased levels, or detection of, the levels of certain molecules (e.g., mRNA, cDNA, or proteins) in a biological sample can be used to identify subjects with a hematological cancer, such as diffuse large B-cell lymphoma (DLBCL) or CLL / SLL, who may be responsive to a therapeutic compound, predict the responsiveness of a subject with, or suspected of having, a hematological cancer, such as DLBCL or CLL / SLL, to a therapeutic compound, or monitor the efficacy of a therapeutic compound in treating a hematological cancer in a subject, where the compound is, for example, Compound 1, or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof; Compound 2, or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof; or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

[0081] 5.1 Definition The term "cancer" as used herein includes, but is not limited to, solid cancers and blood-borne cancers. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell proliferation. Cancer can be cancer of hematopoietic and lymphatic tissues. Hematological cancer refers to cancer that affects the blood, bone marrow, lymph, and lymphatic system.

[0082] The term "diffuse large B-cell lymphoma (DLBCL)" as used herein refers to a neoplasm of medium or large B-lymphoid cells, with a diffuse growth pattern, in which the nuclei of the cells are the same size or larger than those of normal macrophages, or more than twice the size of the nuclei of normal lymphoid cells. DLBCL is a type of non-Hodgkin's lymphoma (NHL), with at least three known subtypes: germinal center B-cell (GCB), activated B-cell (ABC), and primary mediastinal B-cell lymphoma (PMBL). DLBCL cells frequently simultaneously have rearrangements of MYC and / or BCL2 and / or BCL6. For example, in some variants, DLBCL may involve chromosomal changes in the BCL-6 gene at the 3q27 locus, which is important for germinal center formation, and additional rearrangements that affect BCL6. In addition, DLBCL may have gene rearrangements in the immunoglobulin (IG) heavy chain locus, such as t(8;14)(q24;q32) and / or t(14;18)(q32;q21.3), which correspond to MYC, BCL2, or BCL6. When MYC, BCL6, or BCL2 is translocated to the IG locus, it usually results in high levels of mRNA and protein, due to active transcription by the constitutively active IG promoter. Thus, DLBCL cells often have high levels of MYC, BCL6, or BCL2 protein.

[0083] A "subject" or "patient" as used herein is an animal, typically a mammal, including a human, such as a human patient. The term "healthy subject" as used herein is intended to mean an individual who does not have a blood cancer, such as DLBCL or CLL / SLL. An exemplary "healthy subject" has no pre-existing medical conditions. However, it is understood that a "healthy subject" may have a medical condition that is unrelated to a blood cancer, such as diabetes, cardiovascular disease, or any other disease or disorder that does not affect the diagnosis, treatment, biomarker levels, and / or pharmacodynamics of a blood cancer therapy, such as DLBCL or CLL / SLL therapy.

[0084] The term "likely" as used herein generally refers to a high probability of an event. When used in relation to the responsiveness of a patient, the term "likely" generally refers to a high probability that the patient will be responsive to a therapeutic compound. When used in relation to the responsiveness of a therapeutic compound, the term "likely" generally refers to a high probability that the compound will slow the progression of disease or the proliferation of blood cancer cells. When used in relation to the responsiveness of a therapeutic compound, the term "likely" may also generally refer to an increase in indicators such as mRNA or protein that may demonstrate an increase in responsiveness to a therapeutic compound.

[0085] As used herein, the term "successful" or "responsiveness" when used in relation to treatment refers to the degree of effectiveness of the treatment in alleviating or reducing the symptoms of the disease to be treated, such as DLBCL or CLL / SLL.For example, the term "increased responsiveness" when used in relation to the treatment of a cell or subject refers to the increased effectiveness of reducing or reducing the symptoms of the disease, as measured by any method known in the art.In certain embodiments, the increase in effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or more.However, it is also understood that responsiveness can only stop the progression of the disease, and does not necessarily have to alleviate or reduce the symptoms of the disease.

[0086] As used herein, unless otherwise specified, the term "relapse" refers to a disorder, disease or condition that has responded to treatment (e.g., achieved a complete response) and then progressed. Treatment can include one or more lines of therapy. In one embodiment, the disorder, disease or condition has previously been treated with one or more lines of therapy. In another embodiment, the disorder, disease or condition has previously been treated with one, two, three or four lines of therapy. In one embodiment, the disorder, disease or condition is a hematological cancer, such as DLBCL or CLL / SLL.

[0087] As used herein, unless otherwise specified, the term "refractory" refers to a disorder, disease, or condition that has not responded to prior therapy, which may include one or more lines of therapy. In one embodiment, the disorder, disease, or condition has been previously treated with one, two, three, or four lines of therapy. In one embodiment, the disorder, disease, or condition has been previously treated with two or more lines of therapy and has not achieved a complete response (CR) to a regimen that includes the most recent systemic therapy. In one embodiment, the disorder, disease, or condition is a hematological cancer, such as DLBCL or CLL / SLL.

[0088] In one embodiment, "relapsed or refractory" CLL / SLL may refer to CLL / SLL that has been previously treated with one or more lines of therapy. In one embodiment, relapsed or refractory CLL / SLL is CLL / SLL that has been previously treated with one, two, three or four lines of therapy. In one embodiment, relapsed or refractory CLL / SLL is CLL / SLL that has been previously treated with two or more lines of therapy. In one embodiment, relapsed or refractory CLL / SLL is CLL / SLL that has been previously treated with a Bruton's tyrosine kinase (BTK) inhibitor. In one embodiment, relapsed or refractory CLL / SLL has relapsed or is refractory to a BTK inhibitor. In one embodiment, the BTK inhibitor is ibrutinib. In one embodiment, the BTK inhibitor is acalabrutinib. In one embodiment, the BTK inhibitor is zanubrutinib. In one embodiment, the BTK inhibitor is tirabrutinib.

[0089] The term "therapeutic compound" as used herein refers to a compound of formula (I), including compound 1, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; compound 2, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. It should be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be in either the (R) or (S) configuration, or a mixture thereof. It should be recognized that the chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent to administration of the compound in its (S) form for compounds that undergo epimerization in vivo. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthesis or chiral reagents, or resolved using conventional techniques, such as chromatography on chiral stationary phases. In the present description, in the event of any discrepancy between a chemical name and a chemical structural formula, the structural formula will control.

[0090] The term "tautomer" as used herein refers to isomeric forms of a compound that are in equilibrium with one another. The concentration of isomeric forms will depend on and may vary depending on the environment in which the compound is present, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, pyrazole may exhibit the following isomeric forms, which are referred to as tautomers of each other: [ka]

[0091] Unless otherwise specified, when a compound can take the form of alternative tautomers, regioisomers and / or stereoisomers, all alternative isomers are intended to be included within the scope of the subject matter described in the claims. For example, when a compound can have one of two tautomeric forms, both tautomers are intended to be included herein. Thus, the compounds provided herein may be enantiomerically pure or may be a mixture of stereoisomers or diastereomers. As used herein, and unless otherwise specified, the term "enantiomerically pure" refers to a stereoisomerically pure composition of a compound having one chiral center.

[0092] As used herein and unless otherwise specified, the term "stereoisomer" or "stereoisomerically pure" refers to one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. For example, a stereoisomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound comprises about 80% or more by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, about 90% or more by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, about 95% or more by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, or about 97% or more by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound. The compounds may contain chiral centers and may exist as racemates, individual enantiomers or diastereomers, and mixtures thereof. All such isomeric forms, including mixtures thereof, are included within the embodiments provided herein.

[0093] The use of stereomerically pure forms of such compounds, as well as the use of mixtures of those forms, are encompassed by the embodiments provided herein. For example, mixtures containing equal or unequal amounts of enantiomers of a particular compound may be used in the methods and compositions provided herein. These isomers may be asymmetrically synthesized or resolved using standard techniques, such as chiral columns or chiral resolving agents. For example, Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SH, Tables of Resolving Agents and Optical Resolutions p. 268(EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972);Todd, M., Separation Of Enantiomers: Synthetic Methods(Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2014);Toda, F., Enantiomer Separation: Fundamentals and Practical Methods(Springer Science & Business Media, 2007);Subramanian, See G., Chiral Separation Techniques: A Practical Approach (John Wiley & Sons, 2008); Ahuja, S., Chiral Separation Methods for Pharmaceutical and Biotechnological Products (John Wiley & Sons, 2011).

[0094] "Isotopologue" or "isotopic species" as used herein refers to an isotope-enriched compound. The term "isotopically enriched" refers to an atom or compound that has an isotopic composition different from the natural isotopic composition of that atom or compound. Radiolabeled, isotopically enriched compounds are useful as therapeutic agents, e.g., hematological cancer and inflammation therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. Exemplary isotopologues include deuterium, carbon-13, or nitrogen-15 enriched compounds. For example, an isotopologue can be a deuterium-enriched compound, such as compound 1, 2, or 3, in which deuteration occurs at a chiral center.

[0095] It should be noted that in the event of a discrepancy between a depicted structure and a name given to that structure, the depicted structure bears more weight. In addition, if the stereochemistry of a structure or portion of a structure is not shown, for example, with bold or dashed lines, then that structure or portion of the structure is to be interpreted as encompassing all stereoisomers thereof.

[0096] As used herein, unless otherwise specified, the term "pharmaceutical acceptable salts" refers to salts prepared from pharmaceutical non-toxic acids or bases, including inorganic acids and bases, and organic acids and bases. Suitable pharmaceutical acceptable base addition salts of the compounds provided herein include, but are not limited to, metallic salts prepared from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc, or organic salts prepared from lysine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl-glucamine), and procaine. Suitable non-toxic acids include inorganic and organic acids such as, but are not limited to, acetic acid, alginic acid, anthranilic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, formic acid, fumaric acid, furoic acid, galacturonic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, propionic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid. Others are well known in the art, see, e.g., Remington's Pharmaceutical Sciences, 18th eds., Mack Publishing, Easton PA (1990) or Remington: The Science and Practice of Pharmacy, 19th eds., Mack Publishing, Easton PA (1995).

[0097] The term "sample" as used herein typically, but not necessarily, refers to a material or mixture of materials in fluid form that contains one or more components of interest. Exemplary samples are "biological samples" obtained from a biological subject, including samples of biological tissue or fluid origin obtained, arrived at, or collected in vivo or in situ. Biological samples also include samples from areas of a biological subject that contain pre-cancerous or cancerous cells or tissues. Such samples can be, but are not limited to, organs, tissues, and cells isolated from a mammal. Exemplary biological samples include, but are not limited to, cell lysates, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, and the like. Preferred biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, including bone marrow core biopsies, bone marrow aspirates, isolated bone marrow mononuclear cells, circulating tumor cells, and the like.

[0098] A "biological marker" or "biomarker" is a substance whose detection indicates a particular biological state, such as, for example, the presence of a blood cancer. In some embodiments, biomarkers can be measured individually. In other embodiments, several biomarkers can be measured simultaneously.

[0099] In some embodiments, a "biomarker" indicates a change in the level of mRNA expression that can correlate with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. In some embodiments, the biomarker is a nucleic acid, such as an mRNA or cDNA.

[0100] In further embodiments, a "biomarker" indicates a change in the level of expression of a polypeptide or protein that may correlate with the risk or progression of a disease, or with the patient's susceptibility to treatment. In some embodiments, a biomarker may be a polypeptide or protein, or a fragment thereof. The relative levels of a particular protein may be measured by methods known in the art. For example, immunoblots, antibody-based methods such as enzyme-linked immunosorbent assay (ELISA), or other methods may be used.

[0101] The terms "polypeptide" and "protein" as used interchangeably herein refer to a polymer of three or more amino acids in a contiguous sequence linked via peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides, and the like. The term "polypeptide" as used herein can also refer to a peptide. The amino acids that make up a polypeptide can be naturally occurring or synthetic. A polypeptide can be purified from a biological sample. A polypeptide, protein, or peptide also includes modified polypeptides, proteins, and peptides, such as glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.

[0102] The term "level" as used herein refers to the amount, accumulation, or rate of a biomarker molecule. The level refers to, for example, the amount or synthesis rate of messenger RNA (mRNA) encoded by a gene, the amount or synthesis rate of a polypeptide or protein encoded by a gene, or the amount or synthesis rate of a biomolecule accumulated in a cell or biological fluid. The term "level" refers to the absolute amount of a molecule in a sample, or the relative amount of the molecule, measured under steady-state or non-steady-state conditions.

[0103] "Modification of level" refers to the amount, accumulation or rate of biomarker molecules that differs compared to a specific reference.Modification of level can be either increase or decrease depending on the specific biomarker and / or reference used for comparison.For example, the level of a biomarker, such as a protein level, can be said to be modified if its level decreases in a sample after administration of a therapeutic compound compared to an untreated sample.However, the same biomarker can have an increased level of modification, for example, if its reference level is the level at an earlier time point of treated sample.

[0104] The term "reference level" as used herein refers to a reference level of a biomarker used to assess a test level of the biomarker in a sample from an individual. The reference level can be a normal reference level in a sample from a normal subject, or a disease reference level from a diseased subject. The normal reference level is the expression level of the biomarker in a disease-free subject (i.e., a subject without blood cancer). The diseased state reference level is the expression level of the biomarker in a subject with a positive diagnosis for the disease or condition. The reference level can also be a stage-specific reference level. Stage-specific reference level refers to a level of the biomarker characteristic of a certain progression stage of the disease or condition. The reference level can also be the expression level of the biomarker before treatment or at various time points during treatment. For example, the reference level can be the expression level of the biomarker in bone marrow before treatment. In another example, the reference level can be the expression level of the biomarker in blood at several time points during or after treatment.

[0105] As used herein, the term "detectable" when used in reference to a biomarker means that the amount of the biomarker is above a recognition threshold using known techniques for detecting biomolecules, such as immunochemical or histological methods. For example, a level of a biomarker that is detectable using an immunoblot may be a level that is above the background and / or negative control (e.g., no sample) level. Alternatively, for example, a level of a biomarker that is detectable using quantitative RT-PCR (qPCR) may be a level that is detected at a cycle number that is earlier than the cycle number that is detected in a qPCR reaction using a negative control (e.g., water). It is further understood that a detectable level can be a qualitative or quantitative measurement of the presence or concentration of the biomolecule under investigation, and the above-mentioned assays are merely exemplary.

[0106] The term "predict" or "predicting" as used herein generally means to determine or communicate in advance. For example, when used to "predict" responsiveness to treatment, the term "predicting" can mean that the likelihood of responding or not responding to treatment of a blood cancer can be determined initially, before treatment begins, or before the treatment period has substantially progressed.

[0107] The term "monitoring" or "monitoring" as used herein generally refers to supervising, monitoring, regulating, observing, tracking, or investigating an activity. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness in treating a blood cancer in a patient or tumor cell culture. Similarly, the term "monitoring" when used in relation to patient compliance, either individually or in a clinical trial, refers to tracking or making sure that the patient is actually taking the drug being tested as prescribed. Monitoring can be performed, for example, by tracking the expression of mRNA or protein biomarkers.

[0108] The term "efficacy" as used herein refers to the ability to produce a desired or intended result. When used in relation to the efficacy of a therapeutic compound, "efficacy" refers to the reduction or inhibition of the growth or progression of blood cancer, such as DLBCL or CLL / SLL. It also refers to preventing relapse or recurrence of blood cancer, such as DLBCL or CLL / SLL.

[0109] The term "time point" as used herein refers to a time point at which samples are obtained at discrete intervals that are sufficiently separated in time to allow for a response, if one is expected. The time points can be before, during, or after treatment. It is understood that multiple time points can be taken at each stage of the treatment cycle. For example, samples can be obtained one or more months before treatment and again just before treatment, or twice a day during treatment, or before, during, and after treatment. It is understood that the examples provided above are merely illustrative and are not intended to be limiting.

[0110] As used herein, and unless otherwise specified, the terms "therapeutically effective amount" and "effective amount" of a compound refer to an amount sufficient to provide a therapeutic benefit in the treatment, prevention and / or management of a disease, such as DLBCL or CLL / SLL, or to delay or minimize one or more symptoms associated with the disease or disorder being treated. The terms "therapeutically effective amount" and "effective amount" can include an amount that improves overall therapy, reduces or avoids the symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.

[0111] As used herein and unless otherwise indicated, the terms "treat," "treating," and "treatment" refer to alleviating, in whole or in part, a disorder, disease or condition, or one or more symptoms associated with a disorder, disease or condition, or slowing or halting further progression or worsening of those symptoms, or reducing or eradicating the etiology of the disorder, disease or condition itself, e.g., a hematological cancer such as DLBCL or CLL / SLL.

[0112] As used herein and unless otherwise specified, the term "prevent" refers to a method of delaying and / or preventing the onset, recurrence or spread of a disorder, disease or condition, in whole or in part; barring a subject from acquiring a disorder, disease or condition; or reducing the risk that a subject will acquire a disorder, disease or condition.

[0113] As used herein and unless otherwise indicated, the term "manage" includes preventing the recurrence of a particular disease or disorder in a patient who has had the disease or disorder, prolonging the time in remission in a patient who has had the disease or disorder, reducing the mortality rate of the patient, and / or maintaining a reduction in the severity or avoidance of symptoms associated with the disease or condition being managed.

[0114] The terms "near," "about," or "approximately" as used herein refer to a level that is within a close range of the reference level. A biomarker level at or near a reference level may be lower or higher than the reference level, such as within a range of 50% or more of the reference level. One skilled in the art will understand that a biomarker level does not have to be equal to the reference biomarker level, but is considered to be at or near the reference biomarker level if it is within that range. A representative biomarker level at or near the reference sample biomarker level can be within 75-125% of the reference level.

[0115] The term "neutrophil" as used herein refers to a differentiated myeloid cell. Neutrophils are characterized by expression of the surface marker CD11b and the absence or near absence of the surface markers CD34 and CD33 (i.e., CD11b + , CD34 - , and CD33 - Those skilled in the art will appreciate that the expression or lack of expression of a marker need not be absolute. For example, neutrophils express low levels of CD34 and CD34 - Similarly, the cells express moderate but detectable levels of CD11b and may be characterized as CD11b + The expression level can be empirically determined for both the individual and the device used to measure the marker.

[0116] The term "second active agent" as described herein refers to any additional therapeutic agent that is biologically active. It is understood that the second active agent can be a hematopoietic growth factor, a cytokine, an anti-cancer drug, an antibiotic, a cox-2 inhibitor, an immunomodulatory agent, an immunosuppressant, a corticosteroid, a therapeutic antibody that specifically binds to a cancer antigen or a pharmacologically active variant, or a derivative thereof. Exemplary second active agents include, but are not limited to, HDAC inhibitors (e.g., panobinostat, romidepsin, vorinostat, or sitanostat), BCL2 inhibitors (e.g., venetoclax), BTK inhibitors (e.g., ibrutinib or acalabrutinib), mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., idelalisib), PKCβ inhibitors (e.g., enzastaurin), SYK inhibitors (e.g., fostamatinib), JAK2 inhibitors (e.g., ferricyanide), and the like. or momelotinib), Aurora A kinase inhibitors (e.g., alisertib), EZH2 inhibitors (e.g., tazemetostat, GSK126, CPI-1205, 3-deazaneplanocin A, EPZ005687, EI1, UNC1999, or sinefungin), BET inhibitors (e.g., virabresib or 4-[2-(cyclopropylmethoxy)-5-(methylphenyl)-2-propanediol), [tansulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one), hypomethylating agents (e.g., 5-azacytidine or decitabine), chemotherapeutic agents (e.g., bendamustine, doxorubicin, etoposide, methotrexate, cytarabine, vincristine, ifosfamide, melphalan, oxaliplatin, or dexamethasone), or epigenetic compounds (e.g., DOT1L inhibitors such as pinometostat, HAT inhibitors such as C646, and selective inhibitors of leukemia, including leukemia, myeloma, and ovarian cancer. These include selective inhibitors of leukemia, including leukemia, myeloma, and ovarian cancer ...

[0117] The term "symptomatic therapy" as used herein refers to any substance that treats, prevents or manages the side effects of treatment with Compound 1, Compound 2 or Compound 3, or an enantiomer or mixture of enantiomers, tautomers, isotopologues or pharma- ceutically acceptable salts thereof. It is understood that the term "supportive care therapy" refers to any therapeutic agent that is primarily directed at maintaining the strength and / or comfort of the patient. Exemplary supportive care therapies include, but are not limited to, therapies for pain control, intravenous fluids, and electrolyte support, such as isotonic saline, glucose saline, or balanced crystalloid solutions.

[0118] As used herein, the term "source" when used in reference to a reference sample refers to the origin of the sample. For example, a sample taken from blood will also have a reference sample taken from blood. Similarly, a sample taken from bone marrow will also have a reference sample taken from bone marrow.

[0119] The term "expressed" or "expression" as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule that is at least partially complementary to a region of one of the two nucleic acid strands of the gene. The term "expressed" or "expression" as used herein also refers to the transcription from an RNA molecule to give a protein, polypeptide, or portion thereof.

[0120] The terms "cereblon" or "CRBN" and similar terms refer to a polypeptide ("polypeptide", "peptide" and "protein" are used interchangeably herein) that includes the amino acid sequence of any CRBN, such as the human CRBN protein (e.g., human CRBN isoform 1, GenBank Accession No. NP_057386; or human CRBN isoform 2, GenBank Accession No. NP_001166953, each of which is incorporated herein by reference in its entirety), and related polypeptides, including SNP variants thereof. Related CRBN polypeptides include allelic variants (e.g., SNP variants), splice variants, fragments, derivatives, substitution variants, deletion variants, insertion variants, fusion polypeptides, and interspecies homologs, which, in certain embodiments, are sufficient to retain CRBN activity and / or generate an anti-CRBN immune response.

[0121] The term "cereblon-associated protein" or "CAP" as used herein refers to a protein that directly or indirectly interacts with or binds to cereblon (CRBN). For example, the term refers to any protein that directly binds to cereblon, as well as any protein that is an indirect downstream effector in the CRBN pathway. Exemplary CAPs are substrates of CRBN, such as protein substrates of E3 ubiquitin ligase complexes involving CRBN, such as IKZF1, IKZF3, or ZFP91.

[0122] The term "interferon-inducible gene" as used herein refers to a gene whose expression increases in response to type I interferon-mediated signal transduction. For example, binding of interferon (IFN) to type I IFN receptor can trigger the activation of a signal transduction cascade involved in the induction of interferon-inducible genes. Representative genes include interferon regulatory 7 (IRF7), interferon-inducible protein 3 with tetratricopeptide repeats 3 (IFIT3), and dead-box protein 58 (DDX58).

[0123] As used herein, the term "associated with" when used in relation to a signal transduction pathway, a cellular process, or a cellular function shall mean that the molecule is a member of a group of molecules in a cell that act together, for example, to regulate a particular process or function. It is recognized that a molecule can be associated with a signal transduction pathway because it is directly or indirectly involved in driving the transmission of signals, such as interferon signal transduction or cytokine / chemokine signal transduction. A molecule can also be associated with a cellular process or function, such as, for example, cell adhesion, cell-cell junctions, G-protein-coupled receptors, extracellular matrix, cell cycle, or transcription, because the molecule is directly or indirectly involved in a cellular process or function.

[0124] The terms "T cell activation" and "activation of T cells" as used herein shall mean the cellular activation of resting naive T cells into effector T cells capable of inducing tumor cell death. T cell activation may be initiated by the interaction of the T cell receptor (TCR) / CD3 complex with an antigen. Exemplary T cell activation exhibits a cellular response, including, but not limited to, cell proliferation, cytokine secretion, and / or effector function. In the context of this application, T cell activation may be induced by treatment with Compound 1, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; Compound 2, or a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof; or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.

[0125] The term "T cell activation-associated cytokine" as used herein refers to any of a number of factors that are secreted by or whose secretion is increased upon activation of T cells compared to resting, naive T cells. Exemplary T cell activation-associated cytokines include interleukin-2 (IL-2).

[0126] The terms "antibody," "immunoglobulin," or "Ig," as used interchangeably herein, encompass fully assembled antibodies and antibody fragments that retain the ability to specifically bind to an antigen. Antibodies provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, single chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and any epitope-binding fragments as described above. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen-binding domains or molecules that contain an antigen-binding site that immunospecifically binds to a CRBN antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-CRBN antibody). Immunoglobulins may be composed of heavy and light chains. The antibodies provided herein may be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. In some embodiments, the anti-CRBN antibodies are fully human antibodies, such as fully human monoclonal CRBN antibodies. In certain embodiments, the antibodies provided herein are IgG antibodies, or subclasses thereof (e.g., human IgG1 or IgG4). In other embodiments, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen binding domains or molecules that contain an antigen binding site that immunospecifically binds to, for example, Aiolos, Ikaros, c-MYC, IRF4, caspase-3, or any of the biomarkers provided herein.

[0127] As used herein, the terms "immunospecifically bind", "immunospecifically recognize", "specifically bind" and "specifically recognize" are similar terms in the context of antibodies and refer to molecules that bind to an antigen / epitope as understood by those skilled in the art. Antibodies that specifically bind to a target structure or a subunit thereof do not cross-react with biomolecules outside the family of the target structure. In some embodiments, an antibody or antibody fragment binds to a selected antigen in a 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, or 10 -11 Greater than M, 10 -8 M-10 -11 M, 10 -9 M-10 -10 M, and 10 -10 M-10 -11 M. For example, a molecule (e.g., an antibody) that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, e.g., as measured by immunoassays or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen does not cross-react with other proteins.

[0128] The term "epitope" as used herein refers to a localized region on the surface of an antigen that is capable of binding to one or more antigen-binding regions of an antibody, has antigenic or immunogenic activity in an animal, such as a mammal (e.g., human), and is capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody immunospecifically binds, as measured by any method known in the art, for example, by the immunoassays described herein. An antigenic epitope is not necessarily immunogenic. An epitope is usually a surface made up of a group of chemically active molecules, such as amino acids or sugar side chains, and has specific three-dimensional structural characteristics as well as specific charge characteristics. The region of a polypeptide that contributes to an epitope may be contiguous amino acids of the polypeptide, or an epitope may be composed of two or more non-contiguous regions of the polypeptide together. An epitope may or may not have three-dimensional surface characteristics of an antigen.

[0129] The terms "determine," "measure," "assess," "assess," and "assay" as used herein generally refer to any form of measurement and include determining whether an element is present or absent. These terms include quantitative and / or qualitative measurements. The assessment may be relative or absolute. "Assessing the presence of" can include determining the amount of something present, as well as determining whether it is present or absent.

[0130] The term "detectable label" as used herein refers to attaching a specific tag to an antibody to aid in the detection or isolation / purification of protein. Examples of types of labels include, but are not limited to, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), chemiluminescent, enzyme reporters, and elemental particles (e.g., gold particles). Detection can be direct or indirect. Optical methods include microscopy (both confocal and non-confocal), imaging and non-imaging methods. Electrochemical methods include voltammetry and amperometry. Radiofrequency methods include multipolar resonance spectroscopy.

[0131] The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology that are within the skill of those of ordinary skill in the art, and such techniques are fully explained in the literature. Examples of texts that are particularly suitable for reference include: Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); DNA Cloning, vols. I and II, edited by Glover (1985); Oligonucleotide Synthesis, edited by Gait (1984); Nucleic Acid Hybridization, edited by Hames & Higgins (1984); Transcription and Translation, edited by Hames & Higgins (1984); Animal Cell Culture: Immobilized Cells and Enzymes, edited by Freshney (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2nd ed., 1987); and Handbook of Experimental Immunology, editions I-IV, edited by Weir & Blackwell (1986).

[0132] 5.2 Compounds In some embodiments of the various methods provided herein, the compound has formula (I): [ka] or an enantiomer or a mixture of enantiomers, tautomers, isotopologues, or a pharma- ceutically acceptable salt thereof.

[0133] In certain embodiments of the various methods provided herein, the compound is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1): [ka] or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. Methods for making Compound 1 are described in U.S. patent application Ser. No. 16 / 390,815, which is incorporated herein by reference in its entirety.

[0134] In yet another embodiment of the various methods provided herein, the compound is (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 2): [ka] or a tautomer, isotope, or a pharma- ceutically acceptable salt thereof.

[0135] In some embodiments, the compounds are (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione and (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3): [ka] or a mixture of its enantiomers, mixture of enantiomers, tautomers, isotopologues, or a pharma- ceutically acceptable salt thereof.

[0136] Various compounds provided herein may contain chiral centers and may exist as mixtures of enantiomers (e.g., racemic mixtures) or mixtures of diastereomers. Such chiral centers may be in either the (R) or (S) configuration, or may be a mixture thereof. It should be understood that the chiral centers of the compounds provided herein may undergo epimerization in vivo. Thus, one of skill in the art will recognize that administration of a compound in its (R) form is equivalent to administration of a compound in its (S) form for compounds that undergo epimerization in vivo. The methods provided herein encompass the use of stereomerically pure forms of such compounds, as well as mixtures of those forms. For example, mixtures containing equal or unequal amounts of the enantiomers of a particular compound may be used in the methods provided herein. These isomers may be asymmetrically synthesized and resolved using standard techniques, such as chiral columns or chiral resolving agents. See Jacques et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen et al., Tetrahedron 1977, 33:2725-2736; Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0137] Also provided herein are isotopically enriched analogs of the compounds provided herein. Isotopically enriching pharmaceutical agents (e.g., deuteration) to improve pharmacokinetic ("PK"), pharmacodynamic ("PD"), and toxicity profiles has been previously demonstrated for several classes of drugs. See, e.g., Lijinsky et al., Food Cosmet. Toxicol., 20:393 (1982); Lijinsky et al., J. Nat. Cancer Inst., 69:1127 (1982); Mangold et al., Mutation Res. 308:33 (1994); Gordon et al., Drug Metab. Dispos., 15:589 (1987); Zello et al., Metabolism, 43:487 (1994); Gately et al., J Nucl. Med., 27:388 (1986); Wade D, Chem. Biol. Interact. 117:191 (1999).

[0138] Without being limited to a particular theory, isotopic enrichment of a drug may be used, for example, to (1) reduce or eliminate unwanted metabolites, (2) increase the half-life of the parent compound, (3) reduce the number of administrations needed to achieve a desired effect, (4) reduce the dosage needed to achieve a desired effect, (5) increase the formation of any active metabolites, if formed, and / or (6) reduce the production of harmful metabolites in specific tissues, and / or to make a more effective drug and / or a safer drug for use in combination therapy, whether or not the combination therapy is intended.

[0139] When an atom is replaced by one of its isotopes, it often results in a change in the rate of a chemical reaction. This phenomenon is known as the Kinetic Isotope Effect ("KIE"). For example, if a C-H bond is broken during the rate-limiting step of a chemical reaction (the step with the highest transition state energy), the replacement of deuterium with its hydrogen will cause a decrease in the reaction rate and the process will slow down. This phenomenon is known as the Deuterium Kinetic Isotope Effect ("DKIE"). (See, e.g., Foster et al., Adv. Drug Res., Vol. 14, pp. 1-36 (1985); Kushner et al., Can. J. Physiol. Pharmacol., Vol. 77, pp. 79-88 (1999)).

[0140] The magnitude of the DKIE can be expressed as the ratio between the rate of a given reaction in which a C-H bond is broken and the rate of the same reaction in which deuterium is replaced with hydrogen. The DKIE can range from about 1 (no isotope effect) to extremely large values ​​of over 50, meaning that when deuterium is replaced with hydrogen, the reaction can be more than 50 times slower. Without being limited to a particular theory, high DKIE values ​​are the result of an uncertainty principle, and may be due in part to a phenomenon known as tunneling. Tunneling occurs because the mass of the hydrogen atom is so small that a transition state containing a proton can sometimes be formed even in the absence of the required activation energy. Because deuterium is more massive than hydrogen, the probability of this occurring is statistically very low.

[0141] Tritium ("T") is a radioactive isotope of hydrogen used in research, fusion reactors, neutron generators, and radiopharmaceuticals. Tritium is a hydrogen atom with two neutrons in its nucleus and an atomic mass close to 3. It exists in the natural environment in extremely low concentrations, most commonly found as T2O. Tritium decays slowly (half-life = 12.3 years), emitting low-energy beta particles that cannot penetrate the outer layer of human skin. Internal exposure is the main hazard associated with this isotope, but large amounts must be taken in to pose a significant health risk. Compared to deuterium, less tritium needs to be consumed before dangerous levels are reached. Substitution of tritium ("T") for hydrogen results in even stronger bonds than deuterium, imparting a numerically larger isotope effect.

[0142] Similarly, substitution of other elemental isotopes, including but not limited to, substitution of carbon with 13C or 14C, sulfur with 33S, 34S or 36S, nitrogen with 15N, and oxygen with 17O or 18O, will provide similar kinetic isotope effects.

[0143] A variety of enzymes are expressed by the animal's body for the purpose of clearing foreign substances, such as therapeutic agents, from its circulatory system. Examples of such enzymes include cytochrome P450 ("CYP"), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases, which react with these foreign substances and convert them into more polar intermediates or metabolites for renal excretion. Some of the most common metabolic reactions of pharmaceutical compounds involve the oxidation of carbon-hydrogen (CH) bonds to either carbon-oxygen (CO) or carbon-carbon (CC) pi bonds. The resulting metabolites may be stable or unstable under physiological conditions and may have substantially different pharmacokinetic, pharmacodynamic, and acute and long-term toxicity profiles compared to the parent compound. For many drugs, such oxidation is rapid. As a result, these drugs often require multiple or high doses per day.

[0144] Isotopic enrichment at specific positions of the compounds provided herein may produce detectable KIEs that affect the pharmacokinetic, pharmacological, and / or toxicity profiles of the compounds provided herein, compared to similar compounds with natural isotopic composition. In one embodiment, deuterium enrichment is performed at the site of C-H bond cleavage during metabolism.

[0145] Standard physiological, pharmaceutical, and biochemical procedures are available for testing compounds and identifying those having the desired antiproliferative activity.

[0146] Such assays include, for example, biochemical, such as binding assays, radioactive incorporation assays, as well as a variety of cell-based assays.

[0147] Compounds of formula (I) or pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, or racemic mixtures thereof can be prepared by methods known to those skilled in the art, for example, by following the procedures described in U.S. Patent No. 8,518,972 B2, or U.S. Patent Application No. 16 / 390,815, each of which is incorporated herein by reference in its entirety. Exemplary methods for preparing the compounds provided herein are described in Examples 1-3 of Section 6.

[0148] 5.3 Biomarkers and their uses The methods provided herein are based, in part, on the discovery that detectable increases or decreases in certain biomarkers upon treatment with a compound are observed in subjects with hematological cancers, such as DLBCL or CLL / SLL, that are responsive to a given therapeutic agent, such as a compound such as Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof, as described in Section 5.2 above. The levels of these biomarkers can be used to identify or measure the responsiveness of a subject to a therapeutic agent, as well as to facilitate treatment of a subject with hematological cancer. In some embodiments, the levels of the biomarkers allow for predicting the response to a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the hematological cancer is non-Hodgkin's lymphoma. In a particular embodiment, the non-Hodgkin's lymphoma is DLBCL. In further embodiments, the DLBCL is relapsed, refractory, or resistant to conventional therapy. In some embodiments, the hematological cancer is CLL / SLL. In further embodiments, the CLL / SLL is relapsed, refractory, or resistant to conventional therapy.

[0149] As described in the Examples in Section 6 and shown in the Figures, the levels of certain proteins, molecules, mRNA or cellular compositions change in response to treatment with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. These biomarkers include markers of apoptosis, cereblon (CRBN)-related proteins, and interferon-inducible genes. For example, as provided herein, treatment of DLBCL cells sensitive to a compound of formula (I), such as compound 1, compound 2, or compound 3, induced apoptosis, reduced expression of CRBN-binding proteins, and increased expression of interferon-inducible genes compared to untreated cells and non-responsive cells. However, it is understood that in certain circumstances, the biomarkers described herein need not change in level (i.e., increase or decrease). For example, in certain embodiments, the basal expression of a protein prior to administering a dose of a therapeutic compound to a subject may predict sensitivity or responsiveness to treatment with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. Thus, the biomarkers provided herein may be useful for identifying or measuring a subject's responsiveness to treatment and for monitoring the effectiveness of treatment, and may facilitate treatment of a subject with a hematological cancer, such as DLBCL or CLL / SLL.

[0150] In certain embodiments, the biomarkers useful in the methods provided herein are markers of apoptosis. As provided herein, treatment of hematological cancer cells, such as DLBCL or CLL / SLL cells, that are sensitive to a compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, can increase the expression and / or activity of pro-apoptotic proteins and decrease the expression and / or activity of anti-apoptotic proteins, resulting in the induction of apoptosis. Thus, in some embodiments, a change in the level of the biomarker is indicative of the induction of apoptosis. In a specific embodiment, the biomarkers indicative of induction of apoptosis are cleaved caspase 3, cleaved caspase 7, cleaved poly(ADP-ribose) polymerase (PARP), BCL2, survivin, phosphatidylserine (PS) and DNA, Bcl-2-like protein 11 (BIM), interleukin 27 (IL27), tumor necrosis factor (TNF), interleukin 10 (IL10), or combinations thereof. Biomarkers indicative of induction of apoptosis can also detect probes or surrogate markers, such as annexin-V, deep red anthraquinone 7 (DRAQ7), and / or 7-amino-actinomycin D (7-AAD), that bind to endogenous proteins and molecules (e.g., phosphatidylserine, DNA, respectively) in apoptotic cells, the detection of which can distinguish between live, apoptotic, and late apoptotic / dead cells. Thus, in some embodiments, biomarkers indicative of induction of apoptosis include Annexin-V, DRAQ7, 7-AAD, or a combination thereof.

[0151] In some embodiments, the biomarker that indicates induction of apoptosis in the sample is higher than the reference level of the biomarker.For example, in certain embodiments, the reference level is the level of the biomarker before administering the compound, and the higher level of the biomarker that indicates induction of apoptosis after administration compared to the reference level indicates that the treatment is effective.In a specific embodiment, the reference level is the level of the biomarker before administering the compound, and the higher level of cleaved caspase 3, cleaved caspase 7, cleaved poly (ADP-ribose) polymerase (PARP), BCL2, survivin, phosphatidylserine (PS) and DNA, Bcl-2-like protein 11 (BIM), interleukin 27 (IL27), tumor necrosis factor (TNF), interleukin 10 (IL10), annexin-V, DRAQ7, 7-AAD, or combinations thereof in the sample after administration compared to the reference level indicates that the treatment is effective. However, it is understood that the biomarker in the sample does not have to be higher than the reference level of the biomarker.Thus, in some embodiments, the biomarker in the sample is lower than the reference level of the biomarker.For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and the low level of the biomarker indicating induction of apoptosis before the second administration is information for adjusting the dosage or frequency of treatment of the subject with blood cancer.In one embodiment, the blood cancer is DLBCL.In another embodiment, the blood cancer is CLL / SLL.

[0152] In some embodiments, the biomarker useful in the methods provided herein is a CRBN binding protein or a transcriptional target of a CRBN binding protein. As provided herein, the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt, has the ability to bind to CRBN, and CRBN expression is required to mediate the effect of treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt. Thus, in some embodiments, the biomarker is cereblon (CRBN), and if CRBN is detectable in the sample or is higher than the reference level, the subject is diagnosed as likely to be responsive to treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt.

[0153] CRBN is an E3 ubiquitin ligase known to promote the degradation of various substrates, including Ikaros (also known as IKZF1), Aiolos (known as IKZF3), and ZFP91 (see, e.g., U.S. Pat. No. 9,857,359 B2; U.S. Patent Application Nos. 15 / 101,869 and 15 / 518,472, published as U.S. Pat. No. 2017-0242014A1 and U.S. Pat. No. 2016-0313300A1, respectively, each of which is incorporated herein by reference in its entirety). As provided herein, treatment of hematological cancer cells with a compound of formula (I) resulted in the degradation of the CRBN-associated proteins Ikaros, Aiolos, and ZFP91, consistent with the strong anti-proliferative effects of the compound of formula (I). In addition, treatment of hematological cancer cells with a compound of formula (I) resulted in derepression of interferon-inducible genes (ISGs), interferon regulatory factor 7 (IRF7), interferon-inducible protein containing tetratricopeptide repeats 3 (IFIT3), and DExD / H-box helicase (DDX58), as well as critical transcription factors c-Myc / MYC, BCL6, and IRF4. Thus, in some embodiments, the biomarker is CRBN binding protein (CAP) or a transcriptional target of a CRBN binding protein. In a specific embodiment, the CRBN binding protein is Ikaros, Aiolos, or ZFP91. In other embodiments, the transcriptional target of a CRBN binding protein is BCL6, c-MYC, or IRF4.

[0154] In some embodiments, the biomarker is CRBN binding protein (CAP) or a transcriptional target protein of CRBN binding protein, and the biomarker in the sample is lower than the reference level of the biomarker.For example, in certain embodiments, the reference level is the level of the biomarker before administering the compound, and the lower level of CRBN binding protein (CAP) or a transcriptional target protein of CRBN binding protein after administration compared to the reference level indicates the effectiveness of the treatment.In a specific embodiment, the reference level is the level of the biomarker before administering the compound, and the lower level of Ikaros, Aiolos, ZFP91, BCL6, c-MYC, IRF4, or a combination thereof in the sample after administration compared to the reference level indicates the effectiveness of the treatment.However, it is understood that the biomarker in the sample does not have to be lower than the reference level of the biomarker.Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and a high level of the biomarker is information for adjusting the dosage or frequency of treatment of the subject with hematological cancer.In one embodiment, the hematological cancer is DLBCL.In another embodiment, the hematological cancer is CLL / SLL.

[0155] In some embodiments, the biomarker comprises an interferon-inducible gene. In a specific embodiment, the interferon-inducible gene comprises interferon regulatory 7 (IRF7), interferon-inducible protein with tetratricopeptide repeats 3 (IFIT3), DEAD box protein 58 (DDX58), or a combination thereof. In certain embodiments, the biomarker is an interferon-inducible gene, and the biomarker in the sample is higher than the reference level of the biomarker. For example, in certain embodiments, the reference level is the level of the biomarker before administering the compound, and a higher level of the interferon-inducible gene after administration compared to the reference level indicates the effectiveness of the treatment. In a specific embodiment, the reference level is the level of the biomarker before administering the compound, and a higher level of IRF7, IFIT3, DDX58, or a combination thereof after administration compared to the reference level indicates the effectiveness of the treatment. However, it is understood that the biomarker in the sample may not be higher than the reference level of the biomarker. Thus, in some embodiments, the biomarker in the sample is lower than the reference level of the biomarker. For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and a low level of the biomarker is informative for adjusting the dosage or frequency of treatment of the subject with hematological cancer.In one embodiment, the hematological cancer is DLBCL.In another embodiment, the hematological cancer is CLL / SLL.

[0156] As described herein, treatment of hematological cancers with a compound of formula (I) inhibited the proliferation of hematological cancers compared to untreated cells. This was confirmed by an increase in the expression of cyclin-dependent kinase inhibitor 1 (p21), a proliferation inhibitor. Thus, in some embodiments, the biomarker is a marker of proliferation. In a specific embodiment, the biomarker is p21. It is understood that a biomarker can be a marker of increased proliferation and need not be a proliferation inhibitor. By way of example, the marker can be a marker of proliferation (e.g., Brdu, Ki-67, H3pS10, or a similar marker) that is decreased upon treatment with a compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof.

[0157] In some embodiments, the biomarker is a marker of proliferation, and the biomarker in the sample is lower than the reference level of the biomarker. For example, in certain embodiments, the reference level is the level of the biomarker before administration of the compound, and a lower level of the marker of proliferation after administration compared to the reference level indicates the effectiveness of the treatment. In a specific embodiment, the reference level is the level of the biomarker before administration of the compound, and a lower level of the marker of proliferation after administration compared to the reference level indicates the effectiveness of the treatment. However, it is understood that the biomarker in the sample does not have to be lower than the reference level of the biomarker. Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and a higher level of the marker of proliferation is informative for adjusting the dosage or frequency of treatment of a subject with a blood cancer. In one embodiment, the blood cancer is DLBCL. In another embodiment, the blood cancer is CLL / SLL.

[0158] The inventors have observed that the compound of formula (I), or its pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, or racemic mixtures, can also affect non-cancer cells, such as endothelial cells, T and B lymphocytes, fibroblasts, and macrophages. The cells surrounding malignant cells (i.e., tumor microenvironment) can affect malignant cells. For example, anti-inflammatory and immunomodulatory signals can also help treat hematological cancers. Thus, in some embodiments, the biomarker is in a non-cancer cell. In some embodiments, the biomarker is selected from the group consisting of IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, sIL-2, sIL-6, collagen-I and -III, PAI-1, CD69, sIL-10, or a combination thereof. However, it is understood that the biomarker in the sample need not be lower than the reference level of the biomarker. Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.In some embodiments, the biomarker is selected from the group consisting of IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, sIL-2, sIL-6, collagen-I and -III, PAI-1, CD69, sIL-10 or a combination thereof, and the biomarker in the sample is higher than the reference level of the biomarker.For example, in certain embodiments, the reference level is the level of the biomarker before administering the compound, and the lower level of IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, CD-69, collagen-I and -III, PAI-1, or a combination thereof after administration compared to the reference level indicates the effectiveness of treatment. In other embodiments, the reference level is the level of the biomarker before administration of the compound, and a higher level of IL-8, sIL-10, sIL-2, sIL-6, or a combination thereof in the sample after administration compared to the reference level indicates efficacy of the treatment.

[0159] It was observed that compound 1 treatment caused Ikaros / Aiolos-driven upregulation of genes associated with interferon signaling (e.g., IL6ST, IFITM3, IFI6, OAS3, interferon alpha / beta signaling), cytokine / chemokine signaling (e.g., IL23A, CCL1), apoptosis (e.g., IL27, TNF, IL10, caspase), cell adhesion (e.g., SELE, SELPLG, TXA2PA), cell-cell adhesion (e.g., CLDN7, CLDN12), G-protein-coupled receptors (e.g., FFAR2), extracellular matrix (e.g., CD209, SERPINA, SERPINB7), as well as global downregulation associated with cell cycle and transcription. Thus, in some embodiments, the biomarker is associated with interferon signaling. In a specific embodiment, the biomarkers associated with interferon signaling include interleukin-6 signal transducer (IL6ST), interferon-inducible transmembrane protein 3 (IFITM3), interferon alpha-inducible protein 6 (IFI6), 2'-5'-oligoadenylate synthase 3 (OAS3), interferon alpha (IFNα), interferon beta (IFNβ), or a combination thereof. In a specific embodiment, the biomarker is a marker of interferon signaling, and the biomarker in the sample is higher than a reference level of the biomarker after treatment with a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, wherein the reference level is a DMSO-treated sample.

[0160] In other embodiments, the biomarker is associated with cytokine / chemokine signaling. In some embodiments, the biomarker associated with cytokine / chemokine signaling includes interleukin-23 subunit alpha (IL23A), CC motif chemokine 1 (CCL1), or a combination thereof. In a specific embodiment, the biomarker is associated with cytokine / chemokine signaling, and the biomarker in the sample is higher than the reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0161] In further embodiments, the biomarker is associated with cell adhesion.In certain embodiments, the biomarker associated with cell adhesion comprises E-selectin (SELE), P-selectin glycoprotein ligand 1 (SELPLG), thromboxane A2 (TXA2), or a combination thereof.In a specific embodiment, the biomarker is associated with cell adhesion, and the biomarker in the sample is higher than the reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0162] In other embodiments, the biomarker is associated with cell-cell binding. In certain embodiments, the biomarker associated with cell-cell binding comprises laudin 7 (CLDN7), claudin 12 (CLDN12), or a combination thereof. In a specific embodiment, the biomarker is associated with cell-cell binding, and the biomarker in the sample is higher than the reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0163] In some embodiments, the biomarker is a G-protein coupled receptor.In certain embodiments, the G-protein coupled receptor comprises free fatty acid receptor 2 (FFAR2).In a specific embodiment, the biomarker is a G-protein coupled receptor, and the biomarker in the sample is higher than the reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0164] In certain embodiments, biomarker is associated with extracellular matrix.In some embodiments, the biomarker associated with extracellular matrix includes CD209, SERPINA, SERPINB7, or a combination thereof.In a specific embodiment, biomarker is associated with extracellular matrix, and the biomarker in sample is higher than the reference level of biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0165] In other embodiments, the biomarker is associated with the cell cycle.In a specific embodiment, the biomarker is associated with the cell cycle, and the biomarker in the sample is lower than a reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt, wherein the reference level is a DMSO-treated sample.

[0166] In further embodiments, the biomarker is associated with transcription. In a specific embodiment, the biomarker is associated with transcription, and the biomarker in the sample is lower than a reference level of the biomarker after treatment with the compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt, wherein the reference level is the sample treated with DMSO.

[0167] In certain embodiments, the biomarker is the protein level of a protein whose expression changes upon treatment with a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In a specific embodiment, the biomarkers are Aiolos (IKZF3), Ikaros (IKZF1), E3 ubiquitin-protein ligase ZFP91 (ZFP91), protein C-ets-1 (ETS1), Max-binding protein MNT (MNT), myocyte-specific enhancer factor 2B (MEF2B), snRNA activator protein complex subunit 1 (SNAPC1), lysine-specific demethylase 4B (KDM4B), transcription factor AP-4 (TFAP4), nuclear pole transcription factor 1 (UBTF), bromo-adjacent homology domain-containing 1 protein (BAHD1), methyl-CpG-binding domain protein 4 (MBD4), chromobox protein homolog 2 (CBX2), tumor protein 63 (TP63), transducin-like enhancer protein 3 (TLE3), forkhead box protein P1 (FOXP1), zinc finger and BTB domain-containing protein 11 (ZBTB11), interferon-binding protein 1 (IKZF2), and the like. Ron regulatory factor 4 (IRF4), mediator of RNA polymerase II transcription subunit 26 (MED26), cyclic AMP-dependent transcription factor ATF-7 (ATF7), zinc finger protein 644 (ZNF644), lysine-specific demethylase 5B (KDM5B), upstream stimulatory factor 2 (USF2), transcription factor 25 (TCF25), lysine-specific demethylase 4A (KDM4A), lethal (3) malignant brain tumor-like protein 2 (L3MBTL2), nRNA-activated Protein complex subunit 4 (SNAPC4), lysine-specific demethylase 5 (KDM5), transcription factor COE1 (EBF1), forkhead box protein J2 (FOXJ2), nuclear factor of activated T cells cytoplasm 1 (NFATC1), activator of mRNA downregulation protein ZFP36 (ZFP36), hepatocellular carcinoma-inducible growth factor (HDGF), ETS-related transcription factor Elf-1 (ELF1), promyelocytic leukemia protein (PML), and Myb-related protein B.and one or more proteins selected from the group consisting of MYBL2, mother against decapentaplegic homolog 2 (SMAD2), chromodomain-helicase-DNA-binding protein 2 (CHD2), signal transducer and activator of transcription 1 (STAT1), paired-box protein Pax-5 (PAX5), signal transducer and activator of transcription 2 (STAT2), pygopathic homolog 2 (PYGO2), interferon regulatory factor 9 (IRF9), polycomb group RING finger protein 2 (PCGF2), and cyclic AMP-dependent transcription factor ATF-3 (ATF3).

[0168] In some embodiments, the biomarkers include one or more genes selected from the group consisting of interleukin-23 subunit alpha (IL23A), CC motif chemokine 2 (CCL2), and SLIT-ROBO Rho GTPase-activating protein 1 (SRGAP1).

[0169] In certain embodiments, the biomarker comprises a T cell activation marker. Activation of T cells can promote cytotoxic killing of blood cancer cells and thus may be beneficial in the treatment of blood cancer. In certain embodiments, activation of T cells comprises a T cell activation-associated cytokine. In a specific embodiment, the T cell activation-associated cytokine comprises interleukin 2 (IL-2). In some embodiments, the biomarker is a T cell activation marker, and the biomarker in the sample is higher than a reference level of the biomarker. For example, in certain embodiments, the reference level is the level of the biomarker before administration of the compound, and a higher level of the T cell activation marker after administration compared to the reference level indicates the effectiveness of the treatment. In a specific embodiment, the reference level is the level of the biomarker before administration of the compound, and a higher level of IL-2 in the sample after administration compared to the reference level indicates the effectiveness of the treatment. However, it is understood that the biomarker in the sample does not have to be higher than the reference level of the biomarker. Thus, in some embodiments, the biomarker in the sample is lower than the reference level of the biomarker. For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and a low level of the biomarker is informative for adjusting the dosage or frequency of treatment of the subject with hematological cancer.In one embodiment, the hematological cancer is DLBCL.In another embodiment, the hematological cancer is CLL / SLL.

[0170] In other embodiments, the biomarker is a marker for exhausted T cells. Exhausted T cells are phenotypically distinct from functional effector T cells because they acquire expression of inhibitory signaling pathways, including programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 protein (LAG3). Exhausted T cells exhibit reduced differentiation, proliferation, and reduced production of effector cytokines / chemokines (e.g., GM-CSF, TNFα, and IFNγ). Thus, in some embodiments, the biomarker includes PD1, LAG3, or a combination thereof. In further embodiments, the biomarker includes granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), or a combination thereof.

[0171] In some embodiments, the biomarker is a marker of exhausted T cells, and the biomarker in the sample is lower than the reference level of the biomarker.For example, in certain embodiments, the reference level is the level of the biomarker before administering the compound, and a lower level of the marker of exhausted T cells compared to the reference level indicates the effectiveness of the treatment.In a specific embodiment, the reference level is the level of the biomarker before administering the compound, and a lower level of PD1, LAG3 or a combination thereof in the sample after administration compared to the reference level indicates the effectiveness of the treatment.However, it is understood that the biomarker in the sample does not have to be lower than the reference level of the biomarker.Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker.For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, and a higher level of GM-CSF, TNFα, IFNγ or a combination thereof in the sample after administration compared to the reference level indicates the effectiveness of the treatment.

[0172] In some embodiments, the biomarker is a marker of cytotoxicity in non-cancer cells. Neutrophils, as the first cellular component of the inflammatory response and as a key component of innate immunity, represent the first line of defense against infection. Neutropenia blunts the inflammatory response to early infections, allowing bacteriostatic proliferation and infiltration. Neutropenia complications remain the main dose-limiting toxicity of cancer chemotherapy and are associated with significant morbidity and mortality. Thus, ex vivo maturation of neutrophils may be useful for treating hematological cancers, as well as for adjusting the dosage or frequency of treatment of subjects with hematological cancers with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the biomarker is Ikaros expression in neutrophils. In certain embodiments, the biomarker is expressed in white blood cells. In a specific embodiment, the white blood cells include myeloid cells. In a further embodiment, the myeloid cells include neutrophils. In yet a further embodiment, the biomarker is CD11b + , CD34 - , and CD33 - The phenotype of the neutrophils is

[0173] In some embodiments, the biomarker is a marker of cytotoxicity in non-cancer cells, and the biomarker in the sample is lower than the reference level of the biomarker.For example, in certain embodiments, the reference level is the level of the biomarker after the first administration of the compound, and the low level of the marker of cytotoxicity in neutrophils is information for adjusting the dosage or frequency of treatment of the subject of blood cancer.In a specific embodiment, the reference level is the level of the biomarker after the first administration of the compound, and Ikaros and / or CD11b + , CD34 - , and CD33 -A low level of neutrophils having the phenotype is informative for reducing the dosage or frequency of treatment of subjects with blood cancer. However, it is understood that the biomarker in the sample does not have to be lower than the reference level of the biomarker. Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. For example, in some embodiments, the reference level is the level of the biomarker after the first administration of the compound, the reference level is the level of the biomarker after the first administration of the compound, and Ikaros and / or CD11b are both indicated. + , CD34 - , and CD33 - A high level of neutrophils having the phenotype informs the administration of increased dosage or frequency of treatment for a subject with a hematological cancer. In one embodiment, the hematological cancer is DLBCL. In another embodiment, the hematological cancer is CLL / SLL.

[0174] Various target samples can be used for comparison of test samples.For example, non-limiting types of reference samples can be, for example, untreated samples, samples treated at earlier time points during treatment regimen, standardized reference samples, or any other samples suitable for comparison.In some embodiments, the reference biomarker level is the level of the biomarker in a reference sample obtained from a subject before administering a therapeutic compound to the subject, where the reference sample is obtained from the same source as the sample.In other embodiments, the reference biomarker level is the level of the biomarker in a reference sample obtained from a healthy subject without blood cancer, where the reference sample is obtained from the same source as the sample.In yet further embodiments, the reference biomarker level is the level of a predetermined biomarker.

[0175] Those skilled in the art will understand that the alteration of the level of a biomarker will have different interpretations depending on the specific biomarker and the subject sample used for comparison. For example, the level of a biomarker of, for example, apoptosis marker after treatment with a compound of formula (I) or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt may be higher than the reference sample from the subject before administering any therapeutic compound to the subject. An increase in the level of a biomarker may indicate that the treatment is effective. Alternatively, the level of a biomarker of, for example, CRBN binding protein after treatment with a compound of formula (I) or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt may be lower than the reference sample from the subject before administering any therapeutic compound to the subject. A decrease in the level of a biomarker in such an environment may indicate that the treatment is effective.

[0176] However, the same biomarker may have different levels in the same sample when compared with different reference samples.For example, the level of the biomarker, for example, CRBN binding protein, may be higher than the reference sample from the same subject, although the reference sample is obtained at an early time point in the treatment plan, and the level of the biomarker is still lower than the reference sample from the subject before administering any therapeutic compound to the subject, so it still indicates the efficacy of treatment.Therefore, the level of the biomarker and the meaning of the level of the biomarker will depend on the context of the reference sample.

[0177] Thus, in some embodiments, the biomarker in the sample is higher than the reference level of the biomarker. In other embodiments, the biomarker in the sample is lower than the reference level of the biomarker. In yet another embodiment, detection of the biomarker can indicate that the subject is responsive. In certain embodiments, an increase in the level of the biomarker relative to the reference level of the biomarker indicates efficacy of the therapeutic compound in treating hematological cancer in the subject. In other embodiments, a decrease in the level of the biomarker relative to the reference level of the biomarker indicates efficacy of the therapeutic compound in treating hematological cancer in the subject. In one embodiment, the hematological cancer is DLBCL. In another embodiment, the hematological cancer is CLL / SLL.

[0178] The detection of biomarkers is within the skill of a person skilled in the art. For example, in certain embodiments, determining the level of a biomarker comprises determining the protein level of the biomarker. In other embodiments, determining the level of a biomarker comprises determining the mRNA level of the biomarker. In further embodiments, determining the level of a biomarker comprises determining the cDNA of the biomarker as a surrogate marker for determining the RNA level. Exemplary assays provided herein for methods of detecting and quantifying the protein level of biomarkers such as Aiolos, Ikaros, CRBN, c-MYC, IRF4, ZFP91, BCL2, BCL6, MCL1, IRF7, IFIT3, cleaved caspase-3, cleaved caspase-7, cleaved PARP, BIM, DDX58, Survivin, PD1, LAG3, activated T cell associated cytokines, or combinations thereof are immunoassays such as Western blot analysis, enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA), immunohistochemistry (IHC), and fluorescence activated cell sorting (FACS). Exemplary assays provided herein for methods to detect and quantitate RNA levels of biomarkers such as Aiolos, Ikaros, ZFP91, CRBN, c-MYC, IRF4, activated T cell associated cytokine, CD142 (tissue factor), CD62E (E-selectin), interleukin-8 (IL8), interleukin-2 (IL2), interleukin-6 (IL-6), interleukin-17A (IL17A), interleukin-17F (IL17F), collagen-I, collagen-III, PAI-I, interleukin-10 (IL-10), CD69, immunoglobulin (IgG) tumor necrosis factor alpha (TNFα), or combinations thereof are reverse transcription polymerase chain reaction (RT-PCR), e.g., quantitative RT-PCR (qRT-PCR), and RNA-Seq.

[0179] The above techniques for detecting biomarkers are not limiting, and it is understood that one of skill in the art can use any known technique to detect and measure the biomarkers provided herein. Thus, the practice of the embodiments provided herein will utilize, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology that are within the skill of the art. Such techniques are fully explained in the literature.Examples of texts that are particularly suitable for reference include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, eds., DNA Cloning, vols. I and II (1985); Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 1987, 51:263-273; PCR Technology (Stockton Press, NY, Erlich, ed., 1989); Gait et al., Oligonucleotide Synthesis (1984); Hames & Higgins, eds., Nucleic Acid Hybridization (1984); Hames & Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2nd edition 1987); and Weir & Blackwell, eds., Handbook of Experimental Immunology, volumes I-IV (1986).

[0180] 5.4 Treatment and / or management methods Provided herein are methods of treating and / or managing hematological cancers comprising administering a therapeutically effective amount of a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or a pharmaceutically acceptable salt thereof.

[0181] In one aspect, provided herein is a method of selectively treating a hematological cancer in a subject with a hematological cancer, comprising: (a) obtaining a sample from the subject with a hematological cancer; (b) measuring the level of a biomarker in the sample; (c) diagnosing the subject as likely to be responsive to a therapeutic compound if (i) the level of the biomarker in the sample is detectable; or (ii) the level of the biomarker is an altered level compared to a reference level of the biomarker; and (d) administering a therapeutically effective amount of a therapeutic compound to the subject diagnosed as likely to be responsive to the therapeutic compound, wherein the therapeutic compound is a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL). In yet a further embodiment, the DLBCL is relapsed, refractory or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet a further embodiment, the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

[0182] Also provided herein is a method of treating a hematological cancer comprising: (a) obtaining a first sample from a subject with hematological cancer; (b) measuring the level of a biomarker in the first sample; (c) administering a therapeutically effective amount of a therapeutic compound to the subject; (d) obtaining at least one additional sample from the subject after treatment; and (e) measuring the level of the biomarker in the at least one additional sample; and if the level of the biomarker in the at least one additional sample is at or near the level of the biomarker in the first sample, then administering another therapeutically effective amount of a therapeutic compound to the subject, wherein the therapeutic compound is a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is DLBCL. In yet a further embodiment, the DLBCL is relapsed, refractory or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet a further embodiment, the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

[0183] Additionally provided herein is a method of monitoring the effectiveness of a therapeutic compound in treating a hematological cancer in a subject, the method comprising: (a) administering a therapeutic compound to a subject; (b) obtaining a sample from the subject; (c) measuring a level of a biomarker in the sample; and (d) comparing the level of the biomarker in the sample to a reference level of a biomarker, wherein an alteration in the level of the biomarker indicates the effectiveness of the therapeutic compound in treating a hematological cancer in the subject; wherein the therapeutic compound is a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof. In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is DLBCL.In yet further embodiments, the DLBCL is relapsed, refractory or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet further embodiments, the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

[0184] Also provided herein is a method for identifying subjects with blood cancer that may be responsive to therapeutic compounds or predicting the responsiveness of subjects with or suspected of having blood cancer to therapeutic compounds.In some embodiments, a method for identifying subjects with blood cancer that may be responsive to therapeutic compounds or predicting the responsiveness of subjects with or suspected of having blood cancer to therapeutic compounds is provided, comprising: (a) obtaining a sample from the subject; (b) administering the therapeutic compound to the sample; (c) measuring the level of biomarkers in the sample; and (d) diagnosing the subject as being responsive to the therapeutic compound when the level of biomarkers in the sample is altered compared to the level of reference biomarkers; wherein the therapeutic compound is a compound of formula (I), or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is DLBCL. In yet a further embodiment, the DLBCL is relapsed, refractory, or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet a further embodiment, the CLL / SLL is relapsed, refractory, or resistant to conventional therapy.

[0185] In another embodiment, a method for identifying a subject with a hematological cancer that may be responsive to a therapeutic compound or predicting the responsiveness of a subject with or suspected of having a hematological cancer to a therapeutic compound comprises: (a) administering a therapeutic compound to the subject; (b) obtaining a sample from the subject; (c) measuring a level of a biomarker in the sample; and (d) diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is an altered level compared to a reference level of the biomarker; wherein the therapeutic compound is a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; Methods are provided that include a compound selected from the group consisting of indoline-1,3-dione (compound 2), or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (compound 1), or a tautomer, isotopologue, or pharmaceutically acceptable salt thereof. In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a specific embodiment, the non-Hodgkin's lymphoma is DLBCL.In yet further embodiments, the DLBCL is relapsed, refractory or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet further embodiments, the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

[0186] In yet a further embodiment, a method is provided for identifying a subject with a hematological cancer that may be responsive to a therapeutic compound or predicting the responsiveness of a subject with or suspected of having a hematological cancer to a therapeutic compound, comprising: (a) obtaining a sample from the subject; (b) measuring the level of a biomarker in the sample; and (c) diagnosing the subject as likely to be responsive to the therapeutic compound if (i) the level of the biomarker in the sample is detectable; or (ii) the level of the biomarker in the sample is an altered level compared to the level of a reference biomarker; wherein the therapeutic compound is a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt thereof.In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma. In a particular embodiment, the non-Hodgkin's lymphoma is DLBCL. In yet a further embodiment, the DLBCL is relapsed, refractory, or resistant to conventional therapy. In other embodiments, the hematological cancer is CLL / SLL. In yet a further embodiment, the CLL / SLL is relapsed, refractory, or resistant to conventional therapy.

[0187] As described in Section 5.3, various biomarkers can be used in the methods provided herein. In some embodiments, detection or alteration of the level of a biomarker provided herein can be used in a method to identify a subject with a blood cancer that may be responsive to a therapeutic compound. As an example, detection of, for example, CRBN can indicate that a subject with a blood cancer is likely to be responsive. Another exemplary biomarker, such as a CRBN binding protein (e.g., IKAROS, AIOLOS, ZFP91), decreases in response to treatment with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, compared to an untreated sample, and can be used, for example, in a method to identify a subject with a blood cancer that may be responsive to a therapeutic compound, or in a method to predict the responsiveness of a subject with or suspected of having a blood cancer to a therapeutic compound. In other embodiments, detection or alteration of the level of a biomarker provided herein can be used, for example, in a method to treat a blood cancer, or in a method to monitor the efficacy of a therapeutic compound in treating a blood cancer in a patient with a blood cancer. As an example, a therapeutic compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, can be administered to a subject, and the level of a biomarker in a sample from the treated subject can be compared to a reference sample from the same subject before any treatment. For example, an increase in the level of a biomarker of an apoptotic protein can indicate that the treatment is effective in treating the subject, guiding the attending physician. It is understood that the above examples are illustrative and that the biomarkers can not be used comprehensively in the methods provided herein.

[0188] 5.5 How to Adjust Dosage or Dosage Frequency Also provided herein is a method for adjusting the dosage or frequency of administration of a therapeutic compound for treating a subject with hematological cancer, comprising: (a) administering a dosage of the therapeutic compound to the subject; (b) obtaining one or more samples from the subject at different time points; (c) monitoring the level of a biomarker in one or more samples; and (d) adjusting the dose of the therapeutic compound for subsequent administration to the subject based on the altered level of the biomarker in the reference sample; wherein the therapeutic compound is a compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma-ceutically acceptable salt thereof.In certain embodiments, the cycling schedule is determined based on the level of the biomarker. In some embodiments, the compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof; or 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 3), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In certain embodiments, the therapeutic compound of formula (I) is (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1), or a tautomer, isotopologue, or pharma-ceutically acceptable salt thereof. In some embodiments, the hematological cancer comprises non-Hodgkin's lymphoma.In a specific embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma (DLBCL).In yet a further embodiment, the DLBCL is relapsed, refractory or resistant to conventional therapy.In another embodiment, the hematological cancer is CLL / SLL.In yet a further embodiment, the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

[0189] Various biomarkers can be used to determine whether the amount or frequency of administration of treatment needs to be adjusted.In certain embodiments, the biomarker used in the method of adjusting the amount or frequency of administration can be selected from the group consisting of mature neutrophils and the level of Ikaros protein in neutrophils.For example, ex vivo maturation of neutrophils can be used as a biomarker for assessing, for example, myelotoxicity.In certain embodiments, bone marrow CD34 + Ex vivo cultures of cells can be exposed to different treatment regimens with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, e.g., myeloid differentiation can be induced by using stem cell factor (SCF), FMS-related tyrosine kinase 3 ligand (FLT3-L), and granulocyte colony-stimulating factor (G-CSF) in the culture medium. Cell differentiation in the presence or absence of a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt thereof, can result in the differentiation of different populations of cells (e.g., hematopoietic stem cells (HSC, CD34 + / CD33 - / CD11b - ); Stage I cells (CD34 + / CD33 + / CD11b - ); stage II cells (CD134 - / CD33 + / CD11b - ); stage III cells (CD34 - / CD33 + / CD11b + ) and stage IV cells (CD34 - / CD33- / CD11b + (Stage IV) cells) at set time points. In certain embodiments, recovery of mature neutrophils (Stage IV cells) after exposure to a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, can serve as a biomarker.

[0190] In other embodiments, the level of Ikaros protein in neutrophils can be a biomarker for determining dosing regimens and / or cytotoxicity. As provided herein, it has been found that the level of Ikaros in neutrophils decreases during exposure to a compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof, and recovers in a concentration-dependent manner after removal of the drug. The full recovery of late neutrophil maturity is rapidly preceded by the recovery of Ikaros levels. Thus, in some embodiments, the degradation of Ikaros in neutrophils and / or the recovery of Ikaros can be a biomarker for response to a compound of formula (I), or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof, as well as a biomarker for cytotoxicity. In addition, the level of Ikaros protein in neutrophils and / or the maturity of neutrophils can be a biomarker for determining cycle regimens. As an example, recovery of Ikaros protein levels by at least about 30% or more, about 40% or more, about 50% or more, about 60% or more, or about 70% or more following administration of a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, can be used as a biomarker before a subsequent administration of a compound of Formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma-ceutically acceptable salt thereof, is administered.

[0191] It is understood that the biomarkers listed above for use in the methods of adjusting the dosage or frequency of administration of a therapeutic compound for treating a subject with a hematological cancer are not limiting, and other biomarkers indicative of the amount, stage, and / or viability of mature neutrophils may also be useful as biomarkers.

[0192] 5.6 Pharmaceutical Compositions and Routes of Administration As provided herein, the compounds of formula (I) or pharma- ceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, or racemic mixtures thereof, such as any of the compounds described in Section 5.2, may be administered to a subject orally, topically, or parenterally in conventional forms of formulation, such as capsules, microcapsules, tablets, granules, powders, troches, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions, and emulsions. Suitable formulations include excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calcium citrate), lubricants (e.g., magnesium stearate, light anhydrous silicic acid, The pharmaceutical composition may be prepared by a commonly used method using conventional organic or inorganic additives such as talc or sodium lauryl sulfate, flavoring agents (e.g., citric acid, menthol, glycine or orange powder), preservatives (e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben), stabilizers (e.g., citric acid, sodium citrate or acetic acid), suspending agents (e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate), dispersants (e.g., hydroxypropylmethylcellulose), diluents (e.g., water), and base waxes (e.g., cocoa butter, white petrolatum or polyethylene glycol). The effective amount of the compound in the pharmaceutical composition may be a level that will exert the desired effect in a unit dose for both oral and parenteral administration; about 0.001 mg / kg to about 1 mg / kg of subject's body weight.

[0193] The compounds provided herein can be administered orally.In one embodiment, when administered orally, the compounds provided herein are administered with food and water.In another embodiment, the compounds provided herein are dispersed in water or fruit juice (e.g., apple juice or orange juice) and administered orally as a solution or suspension.

[0194] Compounds provided herein may also be administered intradermally, intramuscularly, intraperitoneally, through the skin, intravenously, subcutaneously, intranasally, intraepidermally, sublingually, intracerebrally, intravaginally, transdermally, intrarectally, mucosally, by inhalation, or topically to the ear, nose, eye or skin. The method of administration is left to the discretion of the health care practitioner and may depend in part on the site of the medical condition.

[0195] In one embodiment, provided herein is a capsule containing the compound provided herein without additional carrier, excipient or vehicle.In another embodiment, provided herein is a composition comprising an effective amount of the compound provided herein and a pharma- ceutically acceptable carrier or vehicle, wherein the pharma- ceutically acceptable carrier or vehicle can include excipient, diluent or mixture thereof.In one embodiment, the composition is a pharmaceutical composition.

[0196] The composition can be in the form of tablets, chewable tablets, capsules, liquids, parenteral solutions, troches, suppositories, suspensions, and the like. The composition can be formulated to contain a daily dose, or a convenient portion of a daily dose, in a dosage unit, which can be a single tablet or capsule, or a convenient volume of liquid. In one embodiment, the liquid is prepared from a water-soluble salt. In general, all compositions are prepared according to known methods in pharmaceutical chemistry. Capsules can be prepared by mixing the compound provided herein with a suitable carrier or diluent and filling the appropriate amount of the mixture into capsules. Common carriers and diluents include, but are not limited to, inert powdered substances such as many types of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grains and similar edible powders.

[0197] Tablets can be manufactured by direct compression, wet granulation or dry granulation. In addition to the compound, diluents, binders, lubricants and disintegrants are usually included in these preparations. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders are substances such as starch, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

[0198] In tablet formulations, lubricants may be required to prevent the tablet and punches from sticking in the die. Lubricants may be selected from slippery solids such as talc, magnesium and calcium stearates, stearic acid, and hydrogenated vegetable oils. Tablet disintegrants are substances that swell when wetted with water, causing the tablet to break apart and release the compound. They include starch, clay, cellulose, algins, and gums. More specifically, corn and potato starch, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation exchange resins, alginic acid, guar gum, citrus pulp, and carboxymethylcellulose may be used, as well as sodium lauryl sulfate, for example. Tablets may be coated with sugar as a flavor and sealant, or with a film-forming protecting agent to modify the dissolution properties of the tablet. The composition may also be formulated as a chewable tablet, for example, by using substances such as mannitol in the formulation.

[0199] When it is desired to administer the compound provided herein as suppository, typical base can be used.Cocoa butter is the traditional suppository base, which can be modified by adding wax to slightly raise its melting point.In particular, the water-miscible suppository base, including polyethylene glycols of various molecular weights, is widely used.

[0200] The effect of the compound provided herein can be delayed or extended by appropriate formulation.For example, the slowly solubilizing pellets of the compound provided herein can be manufactured and incorporated into tablets or capsules, or can be manufactured as sustained release implantable devices.This technique also includes manufacturing pellets with several different dissolution rates and filling capsules with a mixture of the pellets.The tablet or capsule can be coated with a film that resists dissolution for a predictable period of time.Even parenteral formulations can be made long-acting by dissolving or suspending the compound provided herein in oily or emulsified vehicles that allow it to disperse slowly in serum.

[0201] The methods provided herein encompass treating patients regardless of their age. In some embodiments, the subject is 18 years of age or older. In other embodiments, the subject is older than 18, 25, 35, 40, 45, 50, 55, 60, 65, or 70 years of age. In other embodiments, the subject is younger than 65 years of age. In other embodiments, the subject is older than 65 years of age.

[0202] Depending on the disease state to be treated and the condition of the subject, Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, CIV, intratesticular injection or infusion, subcutaneous injection, or implantation), inhalation, nasal, vaginal, rectal, sublingual, or topical (e.g., transdermal or topical) routes of administration. Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharmaceutically acceptable salt thereof, may be formulated in an appropriate dosage unit, alone or together with pharmaceutically acceptable excipients, carriers, adjuvants, and vehicles appropriate for each administration route.

[0203] In one embodiment, Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, is administered orally. In another embodiment, Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma-ceutically acceptable salt thereof, is administered parenterally. In yet another embodiment, Compound 1, Compound 2, or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma-ceutically acceptable salt thereof, is administered intravenously.

[0204] Compound 1, compound 2 or compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, can be delivered, for example, as a single bolus injection, or as a single dose, such as an oral capsule, tablet, or pill; or as a time-dependent administration, such as a continuous infusion over time or a bolus administration divided over time. Compounds described herein can be administered repeatedly as necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity.

[0205] Compound 1, compound 2 or compound 3 provided herein, or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt, can be administered once a day (QD) or divided into multiple daily doses, such as twice a day (BID), three times a day (TID) and four times a day (QID). In addition, administration can be continuous (i.e., daily or every day for consecutive days), intermittent, e.g., cyclical (i.e., with drug-free rest for days, weeks or months). The term "daily" as used herein means that therapeutic compounds, such as compound 1, compound 2 or compound 3 provided herein, or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt, are administered once or multiple times a day, e.g., for a period of time. The term "continuous" is intended to mean that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, provided herein, is administered daily for an uninterrupted period of at least 7 days to 52 weeks. The term "intermittent" or "intermittent" as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of Compound 1, Compound 2 or Compound 3, or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, provided herein, can be administered 1-6 days per week, in cycles (e.g., daily administration for 2-8 consecutive weeks followed by a rest period of up to 1 week without administration), or every other day. The term "cycle" as used herein is intended to mean that a therapeutic compound, such as Compound 1, Compound 2 or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, is administered daily or consecutively, but with rest periods.

[0206] In some embodiments, the frequency of administration ranges from approximately daily dose to approximately monthly dose.In one embodiment, administration is once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.In one embodiment, compound 1, compound 2, or compound 3 provided herein, or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt, is administered once a day.In another embodiment, compound 1, compound 2, or compound 3 provided herein, or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharmaceutically acceptable salt, is administered twice a day. In yet another embodiment, Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, is administered three times per day. In yet another embodiment, Compound 1, Compound 2, or Compound 3 provided herein, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, is administered four times per day.

[0207] In one embodiment, the methods provided herein include administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 in one or more 7-day treatment cycles. In another embodiment, the methods provided herein include administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1 through 5 of a 7-day cycle. In one embodiment, Compound 1, Compound 2, or Compound 3 is administered once daily for 5 days, followed by 2 days of rest. In another embodiment, the methods provided herein include administering a therapeutically effective amount of Compound 1, Compound 2, or Compound 3 on days 1 through 5, days 8 through 12, days 15 through 19, and days 22 through 26 of a 28-day cycle.

[0208] In one embodiment, the hematological cancer is chronic lymphocytic leukemia (CLL), and the treatment comprises administering a therapeutically effective amount of a second active agent in one or more treatment cycles. In one embodiment, the second active agent is administered twice every seven days. In one embodiment, the second active agent is administered once every week. In one embodiment, the second active agent is administered once every four weeks. In one embodiment, the second active agent is administered on days 1, 2, 8, and 15 of the first 28-day cycle, and on day 1 of the second through sixth 28-day cycles.

[0209] Any treatment cycle provided herein may be repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more cycles. In certain cases, the treatment cycle as described herein comprises 1 to about 24 cycles, about 2 to about 16 cycles, or about 2 to about 4 cycles. In certain cases, the treatment cycle as described herein comprises 1 to about 4 cycles. In some embodiments, a therapeutically effective amount of Compound 1, Compound 2 or Compound 3, and / or a second active agent is administered in a 28-day cycle for 1 to 24 cycles (e.g., for about 2 years). In certain cases, the cycle therapy is not limited to the number of cycles, and the therapy is continued until disease progression. Cycles may, in certain cases, involve altering the duration of dosing periods and / or rest periods as described herein.

[0210] 5.7 Second Activator The compounds of formula (I), or their enantiomers, mixtures of enantiomers, tautomers, isotopologues, or pharma- ceutically acceptable salts, can be combined with other pharma- ceutically active compounds ("second active agents") in the methods and compositions provided herein. Certain combinations may act synergistically in treating certain types of diseases or disorders, and symptoms and indications associated with such diseases or disorders. The compounds of formula (I), or their enantiomers, mixtures of enantiomers, tautomers, isotopologues, or pharma-ceutically acceptable salts, can also act to alleviate side effects associated with certain second active agents, and vice versa.

[0211] One or more second active ingredients or agents may be used in the methods and compositions provided herein. The second active agent may be a large molecule (e.g., a protein) or a small molecule (e.g., a synthetic inorganic molecule, an organometallic molecule, or an organic molecule). A variety of active agents may be used, such as those described in U.S. patent application Ser. No. 16 / 390,815, filed on even date herewith, or in U.S. Provisional Patent Application entitled "Substituted 4-Aminoisoindoline-1,3-dione Compounds and a Second Active Agent in Combination" (Attorney Docket No. 14247-390-888), each of which is incorporated herein by reference.Exemplary second active agents include, but are not limited to, HDAC inhibitors (e.g., panobinostat, romidepsin, vorinostat, or citalinostat), BCL2 inhibitors (e.g., venetoclax), BTK inhibitors (e.g., ibrutinib or acalabrutinib), mTOR inhibitors (e.g., everolimus), PI3K inhibitors (e.g., idelalisib), PKCβ inhibitors (e.g., enzastaurin), SYK inhibitors (e.g., fostamatinib), JAK2 inhibitors (e.g., , fedratinib, pacritinib, ruxolitinib, baricitinib, gandotinib, lestaurtinib, or momelotinib), Aurora A kinase inhibitors (e.g., alisertib), EZH2 inhibitors (e.g., tazemetostat, GSK126, CPI-1205, 3-deazaneplanocin A, EPZ005687, EI1, UNC1999, or sinefungin), BET inhibitors (e.g., virabresib or 4-[2-(cyclopropylmethoxy) -5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one), hypomethylating agents (e.g., 5-azacytidine or decitabine), chemotherapeutic agents (e.g., bendamustine, doxorubicin, etoposide, methotrexate, cytarabine, vincristine, ifosfamide, melphalan, oxaliplatin, or dexamethasone), or epigenetic compounds (e.g., DOT1L inhibitors such as pinometostat, C646 such as HAT inhibitors, WDR5 inhibitors such as OICR-9429, DNMT1 selective inhibitors such as GSK3484862, LSD-1 inhibitors such as Compound C or secridemstat, G9A inhibitors such as UNC0631, PRMT5 inhibitors such as GSK3326595, BRD inhibitors (e.g., BRD9 / 7 inhibitors such as LP99), SUV420H1 / H2 inhibitors such as A-196, or CARM1 inhibitors such as EZM2302).

[0212] In some embodiments of the methods described herein, the method further comprises administering one or more of rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide, bendamustine (Treanda), lenalidomide, or gemcitabine.In some embodiments of the methods described herein, the treatment further comprises one or more of stem cell transplantation, bendamustine (Treanda) + rituximab, rituximab, lenalidomide + rituximab, or gemcitabine-based combination treatment.In certain embodiments, the second active agent is rituximab, as provided in US provisional patent application 62 / 833,432.

[0213] In one embodiment, the second active agent used in the methods provided herein is a histone deacetylase (HDAC) inhibitor. In one embodiment, the HDAC inhibitor is panobinostat, romidepsin, vorinostat, or citalinostat, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharmaceutically acceptable salt thereof.

[0214] In one embodiment, the HDAC inhibitor is panobinostat, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof. In one embodiment, the HDAC inhibitor is panobinostat. In one embodiment, the HDAC inhibitor is a pharma- ceutically acceptable salt of panobinostat. In one embodiment, the HDAC inhibitor is panobinostat lactate. In one embodiment, the HDAC inhibitor is panobinostat monolactate. The chemical name of panobinostat is (2E)-N-hydroxy-3-[4-({[2-(2-methyl-1H-indol-3-yl)ethyl]amino}methyl)phenyl]acrylamide and has the structural formula: [ka] As shown in the figure.

[0215] In one embodiment, the HDAC inhibitor is romidepsin, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the HDAC inhibitor is romidepsin. The chemical name of romidepsin is (1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-bis(1-methylethyl)-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone, and the structural formula is: [ka] As shown in the figure.

[0216] In one embodiment, the HDAC inhibitor is vorinostat, or a tautomer, isotope, or a pharma- ceutically acceptable salt thereof. In one embodiment, the HDAC inhibitor is vorinostat. Vorinostat's chemical name is N-hydroxy-N'-phenyloctanediamide and has the structural formula: [ka] As shown in the figure.

[0217] In one embodiment, the HDAC inhibitor is an HDAC6 inhibitor. In one embodiment, the HDAC6 inhibitor is citalinostat, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the HDAC6 inhibitor is citalinostat. The chemical name of citalinostat (also known as ACY-241) is 2-((2-chlorophenyl)(phenyl)amino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide, and the structural formula is: [ka] As shown in the figure.

[0218] In one embodiment, the second active agent used in the methods provided herein is a B-cell lymphoma 2 (BCL2) inhibitor. In one embodiment, the BCL2 inhibitor is venetoclax, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the BCL2 inhibitor is venetoclax. The chemical name of venetoclax is 4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) and has the structural formula: [ka] As shown in the figure.

[0219] In one embodiment, the second active agent used in the methods provided herein is a Bruton's tyrosine kinase (BTK) inhibitor. In one embodiment, the BTK inhibitor is ibrutinib, or acalabrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0220] In one embodiment, the BTK inhibitor is ibrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the BTK inhibitor is ibrutinib. The chemical name of ibrutinib is 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one and has the structural formula: [ka] As shown in the figure.

[0221] In one embodiment, the BTK inhibitor is acalabrutinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the BTK inhibitor is acalabrutinib. The chemical name of acalabrutinib is (S)-4-(8-amino-3-(1-(but-2-ynoyl)pyrrolidin-2-yl)imidazo[1,5-a]pyrazin-1-yl)-N-(pyridin-2-yl)benzamide and has the structural formula: [ka] As shown in the figure.

[0222] In one embodiment, the second active agent used in the methods provided herein is a mammalian target of rapamycin (mTOR) inhibitor. In one embodiment, the mTOR inhibitor is also rapamycin or an analog thereof (also referred to as a rapalog). In one embodiment, the mTOR inhibitor is everolimus, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the mTOR inhibitor is everolimus. The chemical name for everolimus is 40-O-(2-hydroxyethyl)-rapamycin and has the structural formula: [ka] As shown in the figure.

[0223] In one embodiment, the second active agent used in the methods provided herein is a phosphoinositide 3-kinase (PI3K) inhibitor. In one embodiment, the PI3K inhibitor is idelalisib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the PI3K inhibitor is idelalisib. The chemical name of idelalisib is 5-fluoro-3-phenyl-2-[(1S)-1-(9H-purin-6-ylamino)propyl]quinazolin-4(3H)-one, and has the structural formula: [ka] As shown in the figure.

[0224] In one embodiment, the second active agent used in the methods provided herein is a protein kinase C beta (PKCβ or PKC-β) inhibitor. In one embodiment, the PKCβ inhibitor is enzastaurin, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the PKCβ inhibitor is enzastaurin. In one embodiment, the PKCβ inhibitor is a pharma- ceutically acceptable salt of enzastaurin. In one embodiment, the PKCβ inhibitor is the hydrochloride salt of enzastaurin. In one embodiment, the PKCβ inhibitor is the bishydrochloride salt of enzastaurin. The chemical name of enzastaurin is 3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione and has the structural formula: [ka] As shown in the figure.

[0225] In one embodiment, the second active agent used in the methods provided herein is a spleen tyrosine kinase (SYK) inhibitor. In one embodiment, the SYK inhibitor is fostamatinib, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the SYK inhibitor is fostamatinib. In one embodiment, the SYK inhibitor is a pharma- ceutically acceptable salt of fostamatinib. In one embodiment, the SYK inhibitor is fostamatinib disodium hexahydrate. The chemical name of fostamatinib is (6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl)methyl dihydrogen phosphate and has the structural formula: [ka] As shown in the figure.

[0226] In one embodiment, the second active agent used in the methods provided herein is a Janus kinase 2 (JAK2) inhibitor. In one embodiment, the JAK2 inhibitor is fedratinib, pacritinib, ruxolitinib, baricitinib, gandotinib, lestaurtinib, or momelotinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0227] In one embodiment, the JAK2 inhibitor is fedratinib, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the JAK2 inhibitor is fedratinib. The chemical name of fedratinib is N-tert-butyl-3-[(5-methyl-2-{4-[2-(pyrrolidin-1-yl)ethoxy]anilino}pyrimidin-4-yl)amino]benzenesulfonamide, and has the structural formula: [ka] As shown in the figure.

[0228] In one embodiment, the JAK2 inhibitor is hapacritinib, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the JAK2 inhibitor is hapacritinib. Pacritinib has the structural formula: [ka] As shown in the figure.

[0229] In one embodiment, the JAK2 inhibitor is ruxolitinib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the JAK2 inhibitor is ruxolitinib. In one embodiment, the JAK2 inhibitor is a pharma- ceutically acceptable salt of ruxolitinib. In one embodiment, the JAK2 inhibitor is ruxolitinib phosphate. The chemical name of ruxolitinib is (R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile and has the structural formula: [ka] As shown in the figure.

[0230] In one embodiment, the second active agent used in the methods provided herein is an Aurora A kinase inhibitor. In one embodiment, the Aurora A kinase inhibitor is alisertib, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the Aurora A kinase inhibitor is alisertib. The chemical name of alisertib is 4-((9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)-2-methoxybenzoic acid and has the structural formula: [ka] As shown in the figure.

[0231] In one embodiment, the second active agent used in the methods provided herein is an enhancer of zest homolog 2 (EZH2) inhibitor. In one embodiment, the EZH2 inhibitor is tazemetostat, GSK126, CPI-1205, 3-deazaneplanocin A (DZNep), EPZ005687, EI1, UNC1999, or sinefungin, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0232] In one embodiment, the EZH2 inhibitor is tazemetostat, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.In one embodiment, the EZH2 inhibitor is tazemetostat.The chemical name of tazemetostat (also known as EPZ-6438) is N-[(1,2-dihydro-4,6-dimethyl-2-oxo-3-pyridinyl)methyl]-5-[ethyl(tetrahydro-2H-pyran-4-yl)amino]-4-methyl-4'-(4-morpholinylmethyl)-[1,1'-biphenyl]-3-carboxamide, and the structural formula is: [ka] As shown in the figure.

[0233] In one embodiment, the EZH2 inhibitor is GSK126, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.In one embodiment, the EZH2 inhibitor is GSK126 (also known as GSK-2816126).The chemical name of GSK126 is (S)-1-(sec-butyl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3-methyl-6-(6-(piperazin-1-yl)pyridin-3-yl)-1H-indole-4-carboxamide, and has the structural formula: [ka] As shown in the figure.

[0234] In one embodiment, the EZH2 inhibitor is CPI-1205, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.In one embodiment, the EZH2 inhibitor is CPI-1205.The chemical name of CPI-1205 is (R)-N-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide, and the structural formula is: [ka] As shown in the figure.

[0235] In one embodiment, the EZH2 inhibitor is 3-deazaneplanocin A. In one embodiment, the EZH2 inhibitor is EPZ005687. In one embodiment, the EZH2 inhibitor is EI1. In one embodiment, the EZH2 inhibitor is UNC1999. In one embodiment, the EZH2 inhibitor is sinefungin.

[0236] In one embodiment, the second active agent used in the methods provided herein is a bromodomain and extra-terminal motif protein (BET) inhibitor. In one embodiment, the BET inhibitor is bilabresib or 4-[2-(cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0237] In one embodiment, the BET inhibitor is bilabresib, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the BET inhibitor is bilabresib. The chemical name of bilabresib (also known as OTX015 or MK-8628) is (S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-hydroxyphenyl)acetamide, and the structural formula is: [ka] As shown in the figure.

[0238] In one embodiment, the BET inhibitor is 4-[2-(cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof. In one embodiment, the BET inhibitor has the structural formula: [ka] As shown in the figure.

[0239] In one embodiment, the second active agent used in the methods provided herein is a hypomethylating agent. In one embodiment, the hypomethylating agent is 5-azacytidine or decitabine, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0240] In one embodiment, the hypomethylating agent is 5-azacytidine, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the hypomethylating agent is 5-azacytidine. The chemical name of 5-azacytidine is 4-amino-l-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one and has the structural formula: [ka] As shown in the figure.

[0241] In one embodiment, the hypomethylating agent is decitabine, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the hypomethylating agent is decitabine. The chemical name of decitabine is 4-amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-one and has the structural formula: [ka] As shown in the figure.

[0242] In certain embodiments, the second active agent used in the methods provided herein is obinutuzumab and the hematological cancer is CLL. In one embodiment, the second active agent comprises a therapeutically effective amount of obinutuzumab administered in one or more treatment cycles. In one embodiment, obinutuzumab is administered twice every 7 days. In one embodiment, obinutuzumab is administered once every week. In one embodiment, obinutuzumab is administered once every 4 weeks. In one embodiment, obinutuzumab is administered on days 1, 2, 8, and 15 of the first 28-day cycle and on the first day of the second through sixth 28-day cycles.

[0243] In one embodiment, obinutuzumab is administered at a dose of about 100 mg on day 1 of the first 28 day cycle, at a dose of about 900 mg on day 2 of the first 28 day cycle, and at a dose of about 1000 mg on each of days 8 and 15 of the first 28 day cycle. In one embodiment, obinutuzumab is administered at a dose of about 1000 mg on days 1 and 2 of the first 28 day cycle, and at a dose of about 1000 mg on each of days 8 and 15 of the first 28 day cycle. In one embodiment, obinutuzumab is administered at a dose of about 1000 mg on day 1 of the second through sixth 28 day cycles.

[0244] In one embodiment, the second active agent used in the methods provided herein is obinutuzumab, and includes administering to a patient a therapeutically effective amount of a compound of Formula (I) in combination with a second active agent provided herein (e.g., venetoclax), further in combination with obinutuzumab.

[0245] In one embodiment, the second active agent used in the methods provided herein is a DOT1L inhibitor. In one embodiment, the DOT1L inhibitor is pinometostat, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the DOT1L inhibitor is pinometostat. Pinometostat (also known as EPZ-5676) has the chemical name (2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3s)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol and has the structural formula: [ka] As shown in the figure.

[0246] In one embodiment, the second active agent used in the methods provided herein is a histone acetyltransferase (HAT) inhibitor. In one embodiment, the HAT inhibitor is C646, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the HAT inhibitor is C646. The chemical name of C646 is 4-(4-((5-(4,5-dimethyl-2-nitrophenyl)furan-2-yl)methylene)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid and has the structural formula: [ka] As shown in the figure.

[0247] In one embodiment, the second active agent used in the methods provided herein is a WD repeat unit-containing protein 5 (WDR5) inhibitor. In one embodiment, the WDR5 inhibitor is OICR-9429, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the WDR5 inhibitor is OICR-9429. The chemical name of OICR-9429 is N-(4-(4-methylpiperazin-1-yl)-3'-(morpholinomethyl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide and has the structural formula: [ka] As shown in the figure.

[0248] In one embodiment, the second active agent used in the methods provided herein is a DNA (cytosine-5)-methyltransferase 1 (DNMT1) inhibitor. In one embodiment, the DNMT1 inhibitor is a DNMT1 selective inhibitor. In one embodiment, the DNMT1 selective inhibitor is GSK3484862, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the DNMT1 selective inhibitor is GSK3484862. The chemical name of GSK3484862 (also known as GSKMI-714) is (R)-2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-phenylacetamide and has the structural formula: [ka] As shown in the figure.

[0249] In one embodiment, the second active agent used in the methods provided herein is a lysine-specific demethylase 1 (LSD-1) inhibitor. In one embodiment, the LDS-1 inhibitor is Compound C or secridemstat, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof.

[0250] In one embodiment, the LSD-1 inhibitor is Compound C, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the LSD-1 inhibitor is Compound C. In one embodiment, the LSD-1 inhibitor is a pharma- ceutically acceptable salt of Compound C. In one embodiment, the LSD-1 inhibitor is Compound C·besylate salt. In one embodiment, the LSD-1 inhibitor is Compound C·monobesylate salt. The chemical name of Compound C is 4-(2-(4-aminopiperidin-1-yl)-5-(3-fluoro-4-methoxyphenyl)-1-methyl-6-oxo-1,6-dihydropyrimidin-4-yl)-2-fluorobenzonitrile and has the structural formula: [ka] As shown in the figure.

[0251] In one embodiment, the LSD-1 inhibitor is seclidemstat, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof. In one embodiment, the LSD-1 inhibitor is seclidemstat. In one embodiment, the LSD-1 inhibitor is a pharma- ceutically acceptable salt of seclidemstat. In one embodiment, the LSD-1 inhibitor is seclidemstat mesylate. The chemical name of seclidemstat (also known as SP-2577) is (E)-N'-(1-(5-chloro-2-hydroxyphenyl)ethylidene)-3-((4-methylpiperazin-1-yl)sulfonyl)benzohydrazide and has the structural formula: [ka] As shown in the figure.

[0252] In one embodiment, the second active agent used in the methods provided herein is a G9A (a histone H3 methyltransferase) inhibitor. In one embodiment, the G9A inhibitor is UNC0631, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the G9A inhibitor is UNC0631. The chemical name of UNC0631 is N-(1-(cyclohexylmethyl)piperidin-4-yl)-2-(4-isopropyl-1,4-diazepan-1-yl)-6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinazolin-4-amine and has the structural formula: [ka] As shown in the figure.

[0253] In one embodiment, the second active agent used in the methods provided herein is a protein arginine methyltransferase 5 (PRMT5) inhibitor. In one embodiment, the PRMT5 inhibitor is GSK3326595, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the PRMT5 inhibitor is GSK3326595. The chemical name of GSK3326595 (also known as EPZ-015938) is (S)-6-((1-acetylpiperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide, and has the structural formula: [ka] As shown in the figure.

[0254] In one embodiment, the second active agent used in the methods provided herein is a bromodomain (BRD) inhibitor. In one embodiment, the BRD inhibitor is a BRD9 / 7 inhibitor. In one embodiment, the BRD9 / 7 inhibitor is LP99, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the BRD9 / 7 inhibitor is LP99. The chemical name of LP99 is N-((2R,3S)-2-(4-chlorophenyl)-1-(1,4-dimethyl-2-oxo-1,2-dihydroquinolin-7-yl)-6-oxopiperidin-3-yl)-2-methylpropane-1-sulfonamide and has the structural formula: [ka] As shown in the figure.

[0255] In one embodiment, the second active agent used in the methods provided herein is an inhibitor of SUV420H1 / H2, two homologous enzymes that methylate lysine 20 of histone H4. In one embodiment, the SUV420H1 / H2 inhibitor is A-196, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the SUV420H1 / H2 inhibitor is A-196. The chemical name of A-196 is 6,7-dichloro-N-cyclopentyl-4-(pyridin-4-yl)phthalazin-1-amine and has the structural formula: [ka] As shown in the figure.

[0256] In one embodiment, the second active agent used in the methods provided herein is a coactivator-associated arginine methyltransferase 1 (CARM1) inhibitor. In one embodiment, the CARM1 inhibitor is EZM2302, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the CARM1 inhibitor is EZM2302. The chemical name of EZM2302 is methyl (R)-2-(2-(2-chloro-5-(2-hydroxy-3-(methylamino)propoxy)phenyl)-6-(3,5-dimethylisoxazol-4-yl)-5-methylpyrimidin-4-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate and has the structural formula: [ka] As shown in the figure.

[0257] In one embodiment, the second active agent used in the methods provided herein is a chemotherapy. In one embodiment, the chemotherapy is bendamustine, doxorubicin, etoposide, methotrexate, cytarabine, vincristine, ifosfamide, melphalan, oxaliplatin, dexamethasone or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, prodrug, or pharmaceutically acceptable salt thereof.

[0258] In one embodiment, the chemotherapy is bendamustine, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is bendamustine. In one embodiment, the chemotherapy is a pharma- ceutically acceptable salt of bendamustine. In one embodiment, the chemotherapy is bendamustine hydrochloride. In one embodiment, the chemotherapy is bendamustine monohydrochloride. The chemical name of bendamustine is 4-(5-(bis(2-chloroethyl)amino)-1-methyl-1H-benzo[d]imidazol-2-yl)butanoic acid and has the structural formula: [ka] As shown in the figure.

[0259] In one embodiment, the chemotherapy is doxorubicin, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is doxorubicin. In one embodiment, the chemotherapy is a pharma- ceutically acceptable salt of doxorubicin. In one embodiment, the chemotherapy is doxorubicin hydrochloride. In one embodiment, the chemotherapy is doxorubicin monohydrochloride. Doxorubicin has the structural formula: [ka] As shown in the figure.

[0260] In one embodiment, the chemotherapy is etoposide, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, prodrug, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is etoposide. Etoposide's chemical name is 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-β-D-glucopyranoside] and has the structural formula: [ka] As shown in the figure.

[0261] In one embodiment, the chemotherapy is a prodrug of etoposide. In one embodiment, the chemotherapy is an ester prodrug of etoposide. In one embodiment, the chemotherapy is etoposide phosphate. The chemical name of etoposide phosphate is 4'-demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-β-D-glucopyranoside],4'-(dihydrogen phosphate) and has the structural formula: [ka] As shown in the figure.

[0262] In one embodiment, the chemotherapy is methotrexate, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is methotrexate. In one embodiment, the chemotherapy is a pharma- ceutically acceptable salt of methotrexate. In one embodiment, the chemotherapy is methotrexate sodium. The chemical name of methotrexate is (4-(((2,4-diaminopteridin-6-yl)methyl)(methyl)amino)benzoyl)-L-glutamic acid and has the structural formula: [ka] As shown in the figure.

[0263] In one embodiment, the chemotherapy is cytarabine, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is cytarabine. The chemical name of cytarabine is 4-amino-1-β-D-arabinofuranosyl-2(1H)pyrimidinone and has the structural formula: [ka] As shown in the figure.

[0264] In one embodiment, the chemotherapy is vincristine, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is vincristine. In one embodiment, the chemotherapy is a pharma- ceutically acceptable salt of vincristine. In one embodiment, the chemotherapy is vincristine sulfate. In one embodiment, the chemotherapy is vincristine monosulfate. Vincristine has the structural formula: [ka] As shown in the figure.

[0265] In one embodiment, the chemotherapy is ifosfamide, or a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is ifosfamide. Ifosfamide's chemical name is 3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxyazaphosphorine 2-oxide and has the structural formula: [ka] As shown in the figure.

[0266] In one embodiment, the chemotherapy is melphalan, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is melphalan. In one embodiment, the chemotherapy is a pharma- ceutically acceptable salt of melphalan. In one embodiment, the chemotherapy is melphalan hydrochloride. In one embodiment, the chemotherapy is melphalan monohydrochloride. The chemical name of melphalan is 4-(bis(2-chloroethyl)amino)-L-phenylalanine and has the structural formula: [ka] As shown in the figure.

[0267] In one embodiment, the chemotherapy is oxaliplatin, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is oxaliplatin. The chemical name of oxaliplatin is cis-[(1R,2R)-1,2 cyclohexanediamine-N,N'][oxalato(2-)-O,O']platinum and has the structural formula: [ka] As shown in the figure.

[0268] In one embodiment, the chemotherapy is dexamethasone, or a stereoisomer, mixture of stereoisomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. In one embodiment, the chemotherapy is dexamethasone. The chemical name for dexamethasone is (11b,16a)-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione, having the structural formula: [ka] As shown in the figure.

[0269] In certain embodiments, the second therapeutic agent is administered before, after, or simultaneously with the compound of formula (I).The administration of the compound of formula (I) and the second therapeutic agent to the patient can occur simultaneously or sequentially, by the same or different administration routes.The suitability of the particular administration route adopted for a particular second drug or agent will vary depending on the second therapeutic agent itself (e.g., whether it can be administered orally or topically without being decomposed before entering the bloodstream) and the subject being treated.The particular administration route of the second drug or agent or ingredient is known to those skilled in the art.See, for example, The Merck Manual, 448 (17th edition, 1999).

[0270] Any combination of the above therapeutic agents suitable for treating a disease or its symptoms may be administered. Such therapeutic agents may be administered with a compound of formula (I), or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, in any combination, either simultaneously or as separate courses of treatment.

[0271] The term "in combination" as used herein does not limit the order in which therapeutic agents (e.g., prophylactic and / or therapeutic agents) are administered to a patient with a disease or disorder.In one embodiment, the first therapy (e.g., a prophylactic or therapeutic agent, such as a compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or its enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt) is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the second therapy (e.g., a second active agent provided herein). In one embodiment, a first therapy (e.g., a compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof) is administered simultaneously with administration of a second therapy (e.g., a second active agent provided herein). In one embodiment, the first therapy (e.g., a prophylactic or therapeutic agent such as a compound provided herein, e.g., Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof) is administered (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) after administration of the second therapy (e.g., a second active agent provided herein).

[0272] Administration to a patient of Compound 1, Compound 2, or Compound 3, or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutically acceptable salt thereof, and a second active agent provided herein may occur simultaneously or sequentially by the same or different routes of administration. The suitability of a particular route of administration employed for a particular active agent will vary depending on the active agent itself (e.g., whether it can be administered orally without being decomposed prior to entering the bloodstream).

[0273] Certain embodiments of the present invention are illustrated by the following non-limiting examples. It is understood that the above detailed description and the following examples are merely illustrative and are not to be taken as limiting the scope of the subject matter of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications (including but not limited to those relating to the compound structures, substituents, derivatives, intermediates, synthesis, formulation and / or methods of use provided herein) can be made without departing from the spirit and scope thereof. U.S. patents and publications cited herein are incorporated by reference.

[0274] 6. Working Example The following examples are carried out using standard techniques that are well known and routine to those skilled in the art, unless otherwise specified in detail, and are intended to be merely illustrative.

[0275] The above embodiments are intended to be merely illustrative, and those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials, and procedures, all of which are considered to be within the scope of the invention and will be covered by the appended claims.

[0276] Example 1: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (compound 3) [ka] To a solution of 4-((4-(chloromethyl)-2-fluorobenzyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (215 mg, 0.500 mmol) (prepared as provided herein) and 4-(azetidin-3-yl)morpholine hydrochloride (107 mg, 0.600 mmol) in dry DMSO (1.7 mL) was added DIEA (262 μL, 1.50 mmol) and the mixture was stirred at ambient temperature for 48 hours. The reaction mixture was diluted with 20% formic acid in DMSO (2.5 mL) and filtered through a membrane syringe filter (0.45 μm nylon). The solution was purified using standard methods to give 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (173 mg, 64.6% yield). LCMS (ESI) m / z 536.2 [M+H] +

[0277] Example 2: Synthesis of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 1) [ka]

[0278] (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-(hydroxymethyl)benzyl)amino)isoindoline-1,3-dione A suspension of (S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5.00 g, 18.3 mmol) and 2-fluoro-4-(hydroxymethyl)benzaldehyde (2.82 g, 18.30 mmol) in 2:1 dioxane-MeOH (75 mL) was cooled to 0° C. and mixed with B 10 H 14(4.92 g, 40.3 mmol) was added in portions over 5 min. The reaction flask was fitted with a septum and needle vent (pressure) and stirred vigorously for 10 min. The mixture was allowed to reach ambient temperature and stirred for 3 h. The mixture was concentrated and the residue was purified by silica gel chromatography (0-10% MeOH-DCM) to give (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-(hydroxymethyl)benzyl)amino)isoindoline-1,3-dione (4.23 g, 56%) as a yellow solid. LCMS (ESI) m / z 411.8[M+H] +

[0279] (S)-4-((4-(chloromethyl)-2-fluorobenzyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0280] A solution of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-(hydroxymethyl)benzyl)amino)isoindoline-1,3-dione (0.727 g, 1.77 mmol) in dry NMP (6 mL) was cooled to 0° C., and methanesulfonyl chloride (0.275 mL, 3.35 mmol) and DIEA (0.617 mL, 3.53 mmol) were added successively. The reaction mixture was allowed to reach ambient temperature and stirred for 18 h. The reaction mixture was slowly added to HO (60 mL) cooled to 0° C. with vigorous mixing. The resulting suspension was filtered, and the solid was collected and washed with HO and EtO. The solid was dissolved in EtOAc and the solution was dried over MgSO4, filtered and concentrated to give (S)-4-((4-(chloromethyl)-2-fluorobenzyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (0.600 g, 79%) as a yellow solid. LCMS (ESI) m / z 430.0 [M+H] +

[0281] (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione

[0282] To a solution of (S)-4-((4-(chloromethyl)-2-fluorobenzyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (300 mg, 0.698 mmol) in dry DMSO (1.0 mL) was added 4-(azetidin-3-yl)morpholine hydrochloride (125 mg, 0.698 mmol) and DIEA (0.122 mL, 0.698 mmol). The reaction mixture was stirred at ambient temperature for 18 h and diluted with DMSO (1 mL). The solution was purified by chiral reverse phase chromatography to give (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (89 mg, 24%, 97% ee). LCMS (ESI) m / z 536.2 [M+H] +

[0283] Example 3: Synthesis of (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (Compound 2) [ka] In addition, the product was subjected to chiral reverse phase chromatography as described in Example 2 to obtain (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione (16 mg, 97% ee). LCMS (ESI) m / z 535.6 [M+H] +

[0284] Example 4: Compound 1 induced apoptosis and inhibited proliferation in diffuse large B-cell lymphoma cell lines The activity of Compound 1 was evaluated in a panel of 23 cell lines of DLBCL. The DLBCL panel includes 7 cell lines classified as activated B cell (ABC) subtype, 13 cell lines classified as germinal center B cell (GCB) subtype, and 3 cell lines classified as primary mediastinal B cell lymphoma (PMBL). In addition, many of these cell lines showed similar cytogenetic features (co-rearrangements of MYC and / or BCL2 or / and BCL6) and molecular features observed in high-risk DLBCL patients. Thus, 11 cell lines were characterized by gene rearrangements of MYC and BCL2 into the immunoglobulin (IG) heavy chain locus, as well as additional rearrangements affecting BCL6. harboring t(8;14)(q24;q32) and / or t(14;18)(q32;q21.3) (Table 1). Translocation of MYC, BCL6, or BCL2 into the IG locus usually results in high levels of mRNA and protein, driven by active transcription by the constitutively active IG promoter. Therefore, protein levels of these genes were monitored by Western blot, confirming overexpression of BCL2, MYC, and / or BCL6 in all cell lines evaluated (Figure 1, Panel A). Expression of CRBN protein and known substrates, Ikaros, Aiolos, and ZFP91, was also evaluated (Figure 1, Panels B and C).

[0285] Table 1: Characteristics of diffuse large B-cell lymphoma cell lines [Table 1] [Table 2] ABC = activated B cell; DLBCL = diffuse large B cell lymphoma; GCB = germinal center B cell; PMBL = primary mediastinal B cell lymphoma; +AMP = gene amplification; ND = no chromosomal rearrangement detected

[0286] Table 2: Antiproliferative and apoptotic effects of compound 1 in diffuse large B-cell lymphoma cell lines. [Table 3] AUC=area under the curve;IC 50 = 50% inhibitor concentration; Emax = maximum effect achieved; NA = not achieved

[0287] A series of cell lines were treated with increasing doses of compound for 5 days, at which point the number of viable cells was measured and the amount of apoptosis determined by 7-AAD exclusion using flow cytometry and Annexin V staining. Table 2 shows that 18 of the 23 lines evaluated had IC50 values ​​for Compound 1 in vitro after 5 days of treatment. 50 Values ​​ranged from 0.001 to 0.4 μM, indicating high sensitivity. Overall, sensitivity to compound 1 was independent of cell of origin (COO), as ABC, GCB, and PMBL subtypes were sensitive to compound 1 (Table 2). In addition, compound 1 showed potent antiproliferative activity in lines harboring MYC, BCL2, and BCL6 chromosomal translocations and / or high protein expression levels for these genes, suggesting the potential for broad activity of compound 1 across a range of DLBCL subtypes (Figure 1; Table 2).

[0288] Dose-response curves showing the loss of viable cells induced by Compound 1 across a range of DLBCL cell lines defined three response categories (Table 2). Ten cell lines had less than 6% viable cells remaining after treatment (E max ) showed high sensitivity to Compound 1; 10 cell lines showed moderate sensitivity with cell viability ranging from 15% to 55%; and 3 cell lines showed limited or no response to Compound 1 (Table 2). To further characterize the antiproliferative effects of Compound 1, Annexin V and 7-AAD staining were measured as indicators of apoptosis. Dramatic induction of apoptosis was observed in the same cell lines identified as sensitive to Compound 1 in the cell proliferation assay (Table 2).

[0289] The results further demonstrated that the antiproliferative effect of Compound 1 did not correlate with absolute levels of baseline cereblon expression: In several cell lines, inhibition of cell proliferation was of similar magnitude even though the expression levels of cereblon protein differed (Figure 1, panels B and C).

[0290] Taken together, these results indicate that Annexin V and 7-AAD staining can be measured as indicators of apoptosis, and that these markers correlate with Compound 1 activity.

[0291] Example 5: Compound 1 inhibited proliferation and induced apoptosis in drug-resistant diffuse large B-cell lymphoma cell lines Compound 1 was tested in cell lines that are resistant to therapeutic agents used in the clinic to treat diffuse large B-cell lymphoma (DLBCL). To this end, the pattern of sensitivity or resistance to compound 1 was compared with doxorubicin, venetoclax, and ibrutinib (three drugs with known activity in DLBCL). The activity of compound 1 was also evaluated in cell lines that had acquired resistance to doxorubicin.

[0292] First, the activity of compound 1 was compared to that of drugs currently used in the treatment of DLBCL by exposing the same set of 23 cell lines (Table 1) to doxorubicin, venetoclax, and ibrutinib. Most cell lines showed clearly distinct response patterns to compound 1, doxorubicin, ibrutinib, and venetoclax (Table 3). All DLBCL cell lines showed strong sensitivity to doxorubicin, including three cell lines that were insensitive to compound 1 (Table 3). Nine of the compound 1-sensitive cell lines were resistant to venetoclax, and ten of the compound 1-sensitive cell lines were resistant to ibrutinib, while four compound 1-sensitive cell lines (SU-DHL-2, Farage, RIVA, WSU-DLBCL2) were resistant to both venetoclax and ibrutinib (Table 3). This indicates that Compound 1 was effective in cell lines that are resistant or refractory to drugs currently used in the treatment of DLBCL, such as venetoclax and ibrutinib.

[0293] Table 3: IC of compound 1, doxorubicin, venetoclax and ibrutinib in diffuse large B-cell lymphoma cell lines 50 concentration [Table 4]

[0294] The activity of compound 1 was also evaluated in cell lines that had acquired resistance to doxorubicin (Table 4). Doxorubicin-resistant cell lines (DoxoR) were generated by culturing the parental cell lines in vitro with increasing concentrations of doxorubicin for extended periods (approximately 9-18 months) until the parental cell lines were able to grow in the presence of relatively high concentrations of doxorubicin (1 μM). Matched parental (M-parental) cells were generated by maintaining the parental cells in culture for the same periods (approximately 9-18 months) without treatment. The growth inhibitory effect of doxorubicin in parental and corresponding resistant cells was evaluated by measuring the number of viable cells and the extent of apoptosis by 7-AAD exclusion and Annexin V staining using flow cytometry. IC for doxorubicin 50 The IC values ​​and cell growth inhibition curves are shown in Table 4. In the DoxoR cell lines, the growth inhibition effect of doxorubicin was greatly reduced compared to its effect in the matched parental cells. The IC values ​​of doxorubicin in the DoxoR lines were 50 The shift in values ​​was greater than 100-fold (Table 4).

[0295] The effect of compound 1 on the viability of doxorubicin-resistant cell lines versus parental cell lines was measured (Figure 2; Table 5). Approximately 20-fold greater sensitivity to compound 1 was measured in resistant OCI-Ly10 cells compared to parental cells (IC 50 IC 50 6 μM), whereas >100-fold greater sensitivity to compound 1 was observed in the doxorubicin-resistant SU-DHL-4 cell line (IC 50 (>10 μM compared to 0.1 μM for

[0296] The increased antiproliferative activity observed for compound 1 in resistant cells, OCI-Ly10 and SU-DHL-4, was accompanied by an increased ability of compound 1 to induce apoptosis. In WSU-DLCL2 cells, the M-parental cell line (IC 50 = 0.01 μM) compared to the DoxoR strain (IC 50=0.1 μM), compound 1 showed better efficacy in the DoxoR cell lines than in the M-parental line, especially at higher concentrations (0.1-1 μM) (high E max In U2932 doxorubicin-resistant cells, a 20-fold lower sensitivity to compound 1 was observed, as well as a lower induction of apoptosis, compared to the M-parental cell line (Figure 2; Table 5).

[0297] Western blot experiments (Figure 3) showed that the increased apoptotic effect of Compound 1 in SU-DHL-4 doxorubicin-resistant cells correlated with the loss of BCL2 expression in this cell line. These results indicate that BCL2 expression can serve as a marker of response to Compound 1, including in drug-resistant cell lines.

[0298] In summary, compound 1 was effective in certain drug-resistant cell lines. Compound 1 showed dose-dependent antiproliferative and cell death responses in all doxorubicin-resistant cell lines tested, indicating that cross-resistance to compound 1 after acquired resistance to doxorubicin may not be common. A lack of cross-resistance between compound 1 and venetoclax or ibrutinib was also demonstrated. Furthermore, markers of apoptosis, such as Annexin V and 7-AAD staining, and protein expression of Bcl-2, can indicate response to compound 1.

[0299] Table 4: Antiproliferative activity of doxorubicin in doxorubicin-resistant and parental cell lines of diffuse large B-cell lymphoma. [Table 5] DoxoR = doxorubicin-resistant lines; IC 50 = 50% inhibitory concentration; M-parent = matched parent

[0300] Table 5: Antiproliferative activity of compound 1 in diffuse large B-cell lymphoma cell lines sensitive and resistant to doxorubicin. [Table 6] DoxoR = doxorubicin-resistant line; E max = Maximum inhibitory response compared to DMSO; IC 50 = 50% inhibitory concentration; M-parent = matched parent

[0301] Example 6: Compound 1 exhibited selective anti-inflammatory, immunomodulatory, and fibrotic and matrix remodeling activities in primary monoculture and coculture systems The activity of Compound 1 was profiled in a series of human primary cell-based assays that model the complex tissue and disease biology of organs (blood vessels, immune system, skin, lungs) as well as general tissue biology using the BioMAP System (DiscoveRx, Fremont, CA).

[0302] The BioMap system consists of 12 human primary monocultures or cocultures under stimulated and unstimulated control conditions (Figure 4, panel 4). Compound 1 mediated changes in key biomarker activities when tested at 0.01, 0.1, 1, and 10 μM. Specifically, the profile for Compound 1 reflected a selective anti-inflammatory and immunomodulatory impact on monocellular (LPS) and T cell-dependent B cell activation responses (BT). Treatment with Compound 1 resulted in a decrease in interleukin 8 (IL-8), interleukin 1a (IL-1a), secreted prostaglandin E2 (sPGE2), and secreted tumor necrosis factor alpha (sTNFα) in the LPS system consisting of peripheral blood mononuclear cells (PBMCs) and endothelial cells. Furthermore, in the BT system, which is composed of B cells and PBMCs, secretory IgG (sIgG), secretory interleukin 17A (sIL-17A), secretory interleukin 17F (sIL-17F), and sTNFα were decreased, whereas secretory interleukin 2 (sIL-2) and secretory interleukin 6 (sIL-6) were increased. In addition, compound 1 was shown to forcibly inhibit collagen-I and -III expression, as well as to slightly inhibit plasminogen activator inhibitor-1 (PAI-1) expression, in the MyoF system, which is composed of lung fibroblasts that model the biology associated with fibrosis and matrix remodeling.

[0303] In conclusion, the profile of compound 1 in the BioMAP Diversity PLUS panel showed that the compound exhibited anti-inflammatory, immunomodulatory, and fibrotic and matrix remodeling activities in primary monoculture and coculture systems, and further demonstrated that the expression of IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, sIL-2, sIL-6, collagen-I and -III, and PAI-1 could serve as biomarkers of response to compound 1 treatment.

[0304] Example 7: Antiproliferative activity of compound 1 is dependent on cereblon Cereblon acts as a substrate receptor for the CRL4 ubiquitin E3 ligase, and binding of cereblon-modulating (CM) compounds induces the recruitment, ubiquitination, and destruction of key target substrates, such as Ikaros, Aiolos, and ZFP91, to mediate cellular effects.

[0305] To determine whether the strong anti-proliferative activity of compound 1 in DLBCL cell lines is dependent on binding to cereblon, we used CRISPR / Cas9 gene editing technology to abolish cereblon expression. Activated B cell (ABC) and MYC / BCL2 dual expressing cell lines SU-DHL-2 and RIVA, germinal center B cell (GCB) and MYC / BCL2 dual hit cell lines (and BCL6 expressing) Karpas-422, SU-DHL-10, WSU-DLCL2, and primary mediastinal B cell lymphoma (PMBL) cell line Farage were infected with a lentiviral construct expressing CAS9 to generate reference CAS9 expressing cells. Cas9 cells were transfected with wild-type cereblon expressing (CRBN WT )

[0306] Cereblon knockout cells (CRBN - / - To generate the CRBN subfamily, individual reference CAS9 cells were infected with a construct expressing a subgenomic single guide RNA (sgRNA). Western blot analysis revealed that CRBN was upregulated relative to the levels of the housekeeping protein β-tubulin. - / - The absence of cereblon protein in cells was confirmed. WT and CRBN - / - Cells were treated with increasing concentrations of Compound 1 for 5 days. Results are shown in Table 1. WT ) is sensitive to compound 1 (IC 50is between 2.5 and 40 nM; Table 6), whereas CRBN knockout blocked the antiproliferative activity of Compound 1 (Table 6), indicating that the antiproliferative effect of Compound 1 on DLBCL cells is cereblon-dependent. These results indicate that CRBN can serve as a biomarker for treatment with Compound 1.

[0307] Table 6: Compound 1, CRBN WT and CRBN - / - Effects of cell proliferation in diffuse large B-cell lymphoma cell lines [Table 7] I C 50 = 50% inhibitory concentration; E max = maximum achievable response

[0308] Example 8: Treatment of diffuse large B-cell lymphoma with Compound 1 caused rapid loss of Aiolos, Ikaros, and ZFP91 and induced apoptosis in vitro. The effect of compound 1 on the intracellular degradation of known cereblon substrates was tested in genetically engineered cell lines. Enzyme fragment complementation (EFC) assays were used to monitor the degradation of DF15 cell lines expressing ePL-tagged target protein substrates of Aiolos, Ikaros, and ZFP91 at concentrations ranging from 6 pM to 10 μM at 1, 2, 6, and 24 hours. Ikaros, Aiolos, and ZFP91 protein levels were measured and assessed (Table 7). After treatment with various concentrations of compound 1 for different times, the remaining ePL-tagged substrates in the cells were measured using a luminescence plate reader. In these assays, compound 1 induced loss of Aiolos (Table 7), loss of Ikaros (Table 7), and loss of ZFP91 (Table 7) in a time- and concentration-dependent manner. Compound 1 was highly potent (low nanomolar or subnanomolar EC ) for the degradation of Aiolos, Ikaros, and ZFP91 at all time points, 1, 2, 6, and 24 h after the compound was added to the cultures. 50However, Compound 1 degraded the three substrates at a concentration as low as 15 nM after 6 h of treatment, with the greatest effect (E max ) was achieved (1%-10% substrate remaining). The degradation kinetics did not change over the 24-h time course, with the maximal effect on degradation persisting until the final time point of 24 h (Table 7).

[0309] Results from cell-based degradation assays using tagged recombinant proteins of known cereblon substrates indicated that compound 1 is a potent and efficient "degrader" of Ikaros, Aiolos, and ZFP91 (Table 7), and these substrates serve as useful biomarkers to assess the activity of compound 1.

[0310] Table 7: Analysis of concentration-response curves for the degradation of substrates at different times of Compound 1 induction [Table 8] EC 50 E = concentration of drug that confers a half-maximal response; max = maximum achievable response; ZFP91 = zinc finger protein 91

[0311] Example 9: Compound 1 promoted the degradation of endogenous substrates in a cereblon-dependent manner and induced apoptosis in DLBCL cell lines To evaluate whether compound 1 promotes the degradation of endogenous substrates in DLBCL cell lines in a cereblon-dependent manner and to demonstrate a potential mechanism of apoptosis induction, time and concentration response studies were performed in the activated B cell (ABC) line, SU-DHL-2 CRBNWT CAS9 control cells, and cereblon knockout SU-DHL2 CRBN- / - Compound 1 was used in cells.

[0312] Treatment with compound 1 at a low dose of 1 nM reduced the expression of SU-DHL-2 CRBNWT In cells, SU-DHL2 caused a time- and concentration-dependent loss of endogenous Ikaros, Aiolos, and ZFP91 proteins. CRBN- / -Cereblon was not lost in DLBCL cells, indicating that cereblon is required for compound 1-induced degradation of endogenous substrates in DLBCL (Figure 5, Panels A, B, and C). Time course analysis revealed that exposure to compound 1 led to rapid degradation of Aiolos, Ikaros, and ZFP91 as early as 4 hours after treatment at all concentrations, and exposure to 10 and 100 nM concentrations of compound 1 led to complete suppression of Ikaros, Aiolos, and ZFP91 protein expression throughout the entire course of treatment. At these two concentrations, in conjunction with the strong suppression of cereblon substrates, compound 1 treatment induced the interferon-stimulated genes IRF7 and IFIT3, reduced the expression of MYC and IRF4, and reduced the expression of apoptotic markers [cleaved caspases 3 and 7 and cleaved poly(ADP-ribose) polymerase (PARP)] as early as 24 hours after treatment (Figure 5, Panels A and B). However, compound 1 did not inhibit the degradation of SU-DHL-2 CRBN- / - The compound had no activity in these cells, indicating that cereblon is required for induction of apoptosis by compound 1 in DLBCL (Figure 5C).

[0313] To confirm the timing of apoptosis induction and its dependence on cereblon, we used a live cell imager that provides kinetic, real-time caspase-3 activation (an indicator of apoptosis) data. CRBNWT Cells were analyzed for caspase-3 activation by following the intracellular cleavage of specific caspase-3 enzymatic substrates over a 112-hour time course. Compound 1-dependent induction of caspase-3 activity began 12 hours after treatment and reached maximum induction at approximately 72 hours (Figure 5D). These results confirm the cereblon-dependent and rapid induction of apoptosis in DLBCL cells by Compound 1 at concentrations of 10 nM and above.

[0314] To confirm that the rapid degradation of substrates and induction of apoptosis by compound 1 is not unique to SU-DHL2 cells, TMD8 (ABC cell line) and Karpus-422 (GCB-cell line) cells were treated with vehicle control (DMSO) or compound 1 for the indicated times and concentrations (Figure 6). Time course analysis reveals that exposure to compound 1 led to rapid degradation of Ikaros and ZFP91 as early as 4 hours after treatment with all concentrations of compound 1. Exposure to 10 and 100 nM compound 1 resulted in complete suppression of Ikaros, Aiolos, and ZFP91 protein expression throughout the entire course of treatment (Figure 6, panels A and C). At these two concentrations, concomitant with strong inhibition of cereblon substrates, compound 1 treatment induced the interferon-stimulated genes DDX58, IRF7, and IFIT3, reduced expression of MYC and IRF4 (only in TMD8, as the GCB strain Karpas-442 does not express IRF4), and induced expression of apoptotic markers as early as 24 h (TMD8) and 48 h (Karpas-422) (Figure 6, panels A and C).

[0315] In a second experiment, we confirmed that treatment of TMD8 with compound 1 (100 nM) resulted in a strong decrease in Aiolos, Ikaros, and ZFP91 at 24 hours. In addition, compound 1 dramatically reduced the abundance of BCL6 (65% decrease), MYC (75% decrease), and IRF4 (90% decrease), and strongly induced cleaved caspase 3 (Figure 6, Panel B), confirming the rapid induction of cell death in TMD8 cells as early as 24 hours (Figure 6, Panel B). In Calpas-422 cells, compound 1 also reduced Aiolos, Ikaros, and ZFP91 at 24 hours (Figure 6, Panel D). At 48 hours, compound 1 increased the abundance of the growth inhibitor p21 and the interferon-stimulated genes IRF7, IFIT3, and DDX58, and reduced the expression of MYC (Figure 6, Panel D). After 72 h, compound 1-treated cells showed decreased levels of MYC (50% reduction), antiapoptotic BCL2, and survivin proteins, as well as robust induction of the apoptotic markers cleaved caspases 3 and 7, and cleaved poly(ADP-ribose) polymerase (PARP) (Figure 6, panel D).

[0316] Taken together, these data indicate that binding of Compound 1 to cereblon results in near-complete degradation of Ikaros, Aiolos, and ZFP91, resulting in a potent and rapid induction of apoptosis in DLBCL cells. Furthermore, these data indicate that expression of cereblon, Ikaros, Aiolos, and ZFP91, interferon-stimulated genes DDX58, IRF7, and IFIT3, apoptosis-related proteins BCL2, survivin, cleaved caspases 3 and 7, and cleaved poly(ADP-ribose)polymerase (PARP), as well as BCL6, MYC, and IRF4, can serve as markers of Compound 1 response.

[0317] Example 10: Multiple cereblon substrates mediated the cytotoxic effects of Compound 1 Compound 1 induces autonomous cell death activity in DLBCL cells in a cereblon-dependent manner. To examine the impact of loss of a single cereblon substrate, we utilized CRISPR / Cas9-mediated knockout of cereblon substrates in combination with a flow cytometry-based cell competition assay to evaluate the relative cell fitness upon gene knockout in six DLBCL cell lines: KARPAS-422, U-2932, RIVA, SU-DHL-16, HT, and SU-DHL-4 (Figure 7, Panel A). To perform the flow cytometry-based competition assay, stable Cas9-expressing DLBCL cells were transduced with a control non-targeting sgRNA construct containing a GFP reporter (sgNT-1-GFP) or with a sgRNA construct targeting a gene of interest containing an RFP reporter (sgRNA-RFP). For targeted knockout of each gene, two sgRNA sequences were used to knockout Ikaros (sgIKZF1-1, sgIKZF1-2), Aiolos (sgIKZF3-1, sgIKZF3-2), and ZFP91 (sgZFP91-1, sgZFP91-3). Controls included two non-targeting sgRNAs (sgNT-1, sgNT-2), an sgRNA targeting a non-coding region of the genome (sgNC-1), and an sgRNA targeting the established essential gene ETF1 (sgETF1-1). After transduction of the sgRNAs, cells were washed and mixed in a 1:1 ratio and incubated with GFP. + and RFP + The percentage of cells was measured every 3 days for 18 days (Figure 7, Panel B). + / RFP + The decrease in the ratio over time indicates that knockout of the gene targeted by the sgRNA in the RFP reporter-containing construct reduces cell fitness. As expected, knockout of the essential gene ETF1 reduced the RFP + / GFP + The second non-targeting control (sgNT-2) behaved very similarly to the first non-targeting control (sgNT-1), resulting in a strong decrease in the proportion of RFP + / GFP+ In addition, a slight decrease in cell fitness was observed in all six DLBCL lines transduced with sgRNA targeting a noncoding region of the genome (sgNC-1), leading to G2 cell cycle arrest, likely due to the induction of double-stranded breaks in DNA.

[0318] Knockout of Ikaros, Aiolos, and ZFP91 induces distinct RFP expression across a series of DLBCL cell lines + / GFP + In addition, knockout of Ikaros, Aiolos, or ZFP91 single genes resulted in a similar degree of RFP1 expression in either cell line as observed with ETF1 knockout. + / GFP + In some cell lines, knockout of Ikaros, Aiolos, or ZFP91 did not result in a decrease in the percentage of RFPs observed. + / GFP + The percentage reduction was similar to that observed with a non-coding sgRNA (sgNC-1), suggesting that gene knockout has the same degree of inhibition in cell fitness as induction of the DNA damage response. Gene knockout in each line was confirmed by immunoblot analysis, revealing that DLBCL cells expressing Cas9 had specific knockouts for the indicated target genes in each of the KARPAS-422-Cas9, U-2932-Cas9, RIVA-Cas9, SU-DHL-16-Cas9, HT-Cas9, and SU-DHL-4-Cas9 cell lines expressing guide RNAs for sgNT-1, sgNT-2, sgNC-1, sgIKZF1-1, sgIKZF1-2, sgIKZF3-1, sgIKZF3-2, sgZFP91-1, sgZFP91-3, and sgETF1-1 cells.

[0319] Overall, these data indicate that the loss of a single cereblon substrate does not fully explain the cytotoxic effects of compound 1, and that the antiproliferative effects may be due to the coordinated loss of multiple cereblon substrates targeted by compound 1. Thus, these results indicate that evaluation of multiple cereblon substrates can be used as a response to compound 1.

[0320] Example 11: Loss of Ikaros and Aiolos sensitized diffuse large B-cell lymphoma cell lines to Compound 1 To examine whether the loss of multiple cereblon substrates targeted by compound 1 may cooperate to cause enhanced inhibition of cell fitness, individual substrates were knocked out and simultaneously treated with increasing concentrations of compound 1. If there is cooperation between substrates to mediate the antiproliferative effect of compound 1, then high loss of any single substrate involved should render cells more sensitive to the compound. A flow cytometry-based cellular competition assay similar to that shown in Figure 7, panel A was utilized to assess enhanced sensitivity to compound 1 with substrate gene knockout. The increased sensitivity to compound 1 due to enhanced loss of a single substrate via gene knockout was shown to be due to the RFP increase in gene knockout cells when treated with compound 1 compared to DMSO. + / GFP + This was indicated by an intensification of the percentage decrease.

[0321] As expected, in control KARPAS-422 (Figure 8, Panel A) and SU-DHL-4 (Figure 8, Panel B) sgNT-1 cells (leftmost group), addition of 0.1 nM, 1 nM, and 10 nM compound 1 (gray bars) did not significantly increase RFP levels compared to DMSO-treated cells (leftmost columns of each subgroup). + / GFP +However, in knockout cells treated with compound 1 for sgIKZF1 (second group from the left; sgIKZF1-1, thin line angled right; and sgIKZF1-2, thick line angled right) and sgIKZF3 (third group from the left; sgIKZF3-1, thin line angled left; and sgIKZF3-2, thick line angled left), RFP was significantly increased compared to their respective DMSO-treated control cells (leftmost row of each subgroup). + / GFP + There was a dose-dependent decrease in the proportion of RFP1 expressed in sgZFP91 cells (far right group; sgZFP91-1, thin check pattern; and sgZFP91-3, thick check pattern), suggesting that loss of ZFP91 does not synergize with loss of other cereblon substrates targeted by compound 1 to enhance the antiproliferative effect of compound 1. In addition, there was a significant decrease in the proportion of RFP1 expressed in DMSO-treated knockout sgIKZF1, sgIKZF3, and sgZFP91 cells (far left black column of each subgroup) over time compared to DMSO-treated reference sgNT-1 cells (far left black column of each subgroup). + / GFP + A decrease in the percentage of substrate degradation was observed, which is a result of decreased cellular fitness resulting from knockout of individual genes, consistent with the data shown in Figure 7, panel B. Furthermore, immunoblot analysis confirmed that treatment with Compound 1 for 24 hours effectively promoted substrate degradation in substrate knockout cells (Figure 9). Thus, these results indicate that Ikaros, Aiolos, and, to a lesser extent, ZFP91, can serve as markers of sensitivity to Compound 1.

[0322] Example 12: Combined loss of Ikaros and Aiolos had an additive effect in inhibiting diffuse large B-cell lymphoma cell fitness Ikaros and Aiolos are highly homologous transcription factors that can form homodimers or heterodimers. To examine whether Ikaros and Aiolos have any redundancy in promoting DLBCL cell survival and proliferation, dual sgRNA-mediated knockout of Ikaros and Aiolos was performed in six DLBCL cell lines: KARPAS-422, U-2932, RIVA, SU-DHL-16, HT, and SU-DHL-4. A flow cytometry-based competition assay was used to assess cell fitness: cells were transduced with sgNT-1-GFP or a construct expressing two sgRNAs from the same vector with an RFP reporter (Figure 10, panel A). The binary sgRNA combinations were sgNT-1+sgNT-2, sgIKZF1-1+sgNT-1, sgIKZF1-1+sgNT-2, sgIKZF3-1+sgNT-1, sgIKZF3-1+sgNT-2, sgIKZF1-1+sgIKZF3-1, and sgIKZF1-2+sgIKZF3-2. Once again, after transduction, cells were washed and mixed in a 1:1 ratio to express GFP+ and RFP + The cell percentages were determined every 3 days for 15 days.

[0323] In all six cell lines, knockout of both Ikaros and Aiolos significantly reduced RFP expression compared with knockout of either Ikaros or Aiolos alone. + / GFP +The specificity for knockout of Ikaros, Aiolos, or both was confirmed by immunoblot analysis, and DLBCL cells expressing Cas9 expressed dual guide RNAs for sgIKZF1-1+sgNT-1, sgIKZF1-1+sgNT-2, sgIKZF3-1+sgNT-1, sgIKZF3-1+sgNT-2, sgIKZF1-1+sgIKZF3-1, and sgIKZF1-2+sgIKZF3-2, KARP We found that in the AS-422-Cas9, U-2932-Cas9, RIVA-Cas9, SU-DHL-16-Cas9, HT-Cas9, and SU-DHL-4-Cas9 cell lines, each of which has a specific knockout for IKZF1 or IKZF3 in cell lines containing the relevant gRNA for the gene, sgNT-1+sgNT-2 showed no change in the levels of IKZF1 or IKZF3 protein.

[0324] These results indicated that Ikaros and Aiolos have some redundant functions in DLBCL cell lines and that loss or degradation of both Ikaros and Aiolos may serve as a marker of response to Compound 1.

[0325] Example 13: Inhibition of Ikaros or Aiolos Degradation Protected Diffuse Large B-Cell Lymphoma Cells from Compound 1 To test whether Ikaros and Aiolos were redundant in promoting survival and proliferation of DLBCL cell lines, degradation-resistant mutants of Ikaros (IKZF1-G151A), Aiolos (IKZF3-G152A), and ZFP91 (ZFP91-G405A) were ectopically and stably expressed in four DLBCL cell lines: KARPAS-422, RIVA, HT, and SU-DHL-4, along with Nano Luc luciferase (Nluc) as a control. Dose-response curves with Compound 1 show that expression of degradation-resistant mutants of Ikaros or Aiolos provided protection from Compound 1 in all four cell lines (Figure 11). In addition, ectopic expression of degradation-resistant mutants, as well as their protection against Compound 1-induced degradation, was confirmed by immunoblot analysis. For example, immunoblot analysis showed that Ikaros, Aiolos, and ZFP91 protein levels were not substantially affected by Compound 1 treatment in KARPAS-422, RIVA, HT, and SU-DHL-4 cell lines ectopically expressing Nluc, compared to the same cell lines ectopically expressing degradation-resistant mutants of Ikaros (IKZF1-G151A), Aiolos (IKZF3-G152A), or ZFP91 (ZFP91-G405A). For example, treatment of Nluc-expressing cells with 5 nM or 50 nM Compound 1 resulted in a significant decrease in IKZF1, IKZF3, and ZFP91 protein levels when compared to control or DMSO-treated cells. Beta-actin levels were used as a control. Ectopic expression of IKZF1-G151A in the four cell lines revealed that IKZF1 protein levels were not decreased in response to treatment with 5 nM or 50 nM compound 1 for 24 h, whereas IKZF3 and ZFP91 levels were still decreased in response to compound 1 treatment. A similar pattern was observed with IKZF3-G152A, and ZFP91-G405A. However, the degree of protection observed in the four cell lines varied. Near-complete protection against compound 1 was observed in KARPAS-422, and SU-DHL-4, suggesting that Ikaros and Aiolos have truly redundant functions in these lines.However, protection was less robust in HT and RIVA. Taken together, these data highlight the utility of responding to Ikaros, Aiolos, or both Compound 1.

[0326] Example 14: Compound 1 promoted the degradation of Ikaros in T cells without significantly affecting their viability The immunomodulatory activity of compound 1 was evaluated in ex vivo stimulated PBMCs. Disease progression in lymphoma patients is associated with impaired immune system function. Exhausted T cells show impaired function in differentiation, proliferation and cytokine production.

[0327] Purified PBMCs from four healthy donors were plated on anti-CD3-coated plates to stimulate T cells and then treated with either DMSO (control) or compound 1 at various concentrations. The ability of compound 1 to promote the degradation of Ikaros was assessed over time by flow cytometry (Figure 12). Compound 1 induced a concentration-dependent degradation of Ikaros in PBMCs, as evidenced by a decrease in the percentage of cells that were positive for Ikaros over time. Moreover, the degradation was maintained immediately after a single treatment of compound 1 for up to 7 days.

[0328] To establish that this was due to the compound's effect of promoting Ikaros degradation and not due to a lack of cell proliferation or viability of CD-3 stimulated PBMCs, the effect of Compound 1 on their viability was assessed by flow cytometry. Compound 1-treated cells showed no significant effect on PBMC viability, which was comparable to the viability of control DMSO-treated cells over time. Peripheral blood mononuclear cells from four healthy donors were exposed to Compound 1 at concentrations of 0.1, 1, 10, or 100 nM for 3, 4, or 7 days, and viability was measured by staining with Viobility Fixable Dye. Results showed that on days 3 and 4, approximately 80% of PBMCs were viable, with Compound 1 having no significant effect at any concentration. The trend in cell viability continued through day 7, with Compound 1 treatment having no effect on PBMC viability.

[0329] These results demonstrate that the expression levels of Ikaros in T cells can be degraded by Compound 1, and that the levels of Ikaros can serve as a marker of response to Compound 1 in T cells.

[0330] Example 15: Effects of Compound 1 on effector lymphocytes and cytokine production The results from Example 14 show that Compound 1 promotes Ikaros degradation, and Ikaros is known to act as a suppressor of IL-2 expression and secretion, a T cell activation marker. Therefore, the effect of Compound 1 on effector T cell cytokine secretion was evaluated.

[0331] Purified PBMCs from four healthy donors were plated on anti-CD3 antibody-coated plates to stimulate T cells and then treated with either DMSO (control) or various concentrations of compound 1 for 3, 4, and 7 days. Using a mesoscale (MSD) assay, cytokine secretion in the supernatant from PBMC cultures was measured over time. Interleukin-2 secretion by PBMCs during in vitro culture increased with exposure to compound 1, as shown in Figures 13 and 14. Compound 1 induced IL-2 secretion at all concentrations tested. This activity with IL-2 occurred at concentrations of compound 1 (0.1-100 nM) that were known to produce strong antiproliferative activity against DLBCL tumor cells. Transient or no effects were observed with other monitored cytokines (TNFα, IFNγ, IL-4, IL-13, IL-10, and IL-6). This demonstrates that IL-2 secretion can serve as a marker of the response to Compound 1, and in particular as a marker for T cell activation.

[0332] Example 16: Compound 1 induced cytokine / chemokine secretion in exhausted T cells Exhausted T cells are phenotypically distinct from functional effector T cells because they acquire expression of inhibitory signaling pathways, including programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 protein (LAG3). Exhausted T cells exhibit reduced differentiation, proliferation, and cytokine production.

[0333] To induce T cell exhaustion, PBMCs from three donors were treated with 100 ng / mL Staphylococcal Enterotoxin B (SEB) for 72 hours (Figure 15, Panels A and C). On day 3, SEB was washed out and expression of exhaustion markers, PD1 and LAG3 on CD-3 positive T cells was assayed by FACS (Figure 15, Panel B). Exhausted T cells from one donor were then subsequently treated with Compound 1 for 96 hours, and exhausted T cells from two additional donors were treated with Compound 1 in the presence of 1 ng / mL SEB for 48 and 96 hours, and release of effector cytokines into the medium was assessed by MSD analysis. Results showed that compound 1 increased the secretion levels of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) in a concentration- and time-dependent manner, as measured by mesoscale (MSD) analysis after 48 or 96 hours. For example, about 0.001 μM compound 1 resulted in 3000 pg / mL of GM-CSF at 96 hours, whereas about 0.01 μM compound 1 resulted in 10,000 pg / mL of GM-CSF at 96 hours, and the secretion continued to increase until it plateaued just below 15,000 pg / mL at a higher concentration than 0.1 μM compound 1. Exposure to compound 1 for 96 hours showed that 96 hours of treatment with compound 1 resulted in the release of higher amounts of GM-CSF and TNFα, and to a lesser extent INFγ, compared to 48 hours of exposure.

[0334] Compound 1 induced the secretion of the effector cytokines / chemokines GM-CSF, TNFα, and IFNγ in a T cell restimulation assay. Compound 1 also demonstrated activity in inducing secretion of the three effector cytokines / chemokines in the three donor PBMCs evaluated at concentrations ranging from 0.01 to 10 μM. GM-CSF levels (EC 50 =0.006 μM), TNFα (EC 50 = 0.01 μM), and IFNγ (EC 50 = 0.01 μM) was achieved. 50supports that the immunomodulatory activity of compound 1 occurs at concentrations that have potent anticancer effects in vitro.

[0335] Taken together, these results demonstrate that compound 1 has immunomodulatory activity. Of note, this occurs at similar concentrations to produce antitumor effects in a range of DLBCL cell lines. Furthermore, these results demonstrate that the secretion of effector cytokines / chemokines GM-CSF, TNFα and IFNγ from T cells can serve as markers of response to compound 1, and that the expression of PD-1 and LAG3 can be used as markers to identify cells that may respond to compound 1 treatment.

[0336] Example 17: Compound 1 and its R-enantiomer exhibit similar antiproliferative activity and bind to cereblon To evaluate the activity of both Compound 1 and Compound 2 in DLBCL cells, CellTiter-Glo (CTG) assay was performed to measure anti-proliferative activity. Potent anti-proliferative activity was observed for both Compound 2 and 3 in DLBCL cell line SU-DHL-4 (Table 8).

[0337] Table 8: Enantiomers were evaluated for antiproliferative potency in diffuse large B-cell lymphoma cells in a 5-day assay. [Table 9] DLBCL=diffuse large B-cell lymphoma; IC 50 = 50% inhibitory concentration

[0338] To investigate whether compound 1 and its enantiomer compound 2 can bind to cereblon, a short-term (10-20 min) ligand competition assay was used. Samples of compound 1 and compound 2 with chiral purity greater than 99% were evaluated for cereblon binding affinity in a TR-fluorescence resonance energy transfer (FRET) assay (Table 8). The results revealed that both compound 1 and compound 2 bind to CRBN in a concentration-dependent manner. Specifically, compound 1 (IC 50 = 0.86 ± 0.1 μM, n = 5) was inhibited by compound 2 (IC 50 = 10.25 ± 1 μM, n = 5).

[0339] Taken together, these results demonstrated that both Compound 1 and Compound 2 exhibit activity in DLBCL cells and that cereblon may serve as a marker of response.

[0340] Example 18: Compound 1 and Compound 2 induce degradation of Aiolos, Ikaros, and ZFP91 Pure samples of each enantiomer were tested for intracellular degradation of substrate proteins measured over a short time course (Figure 16). SU-DHL-2 cells were treated with vehicle control (0.1% DMSO), compound 1 (1, 10, 100 nM), or compound 2 (1, 10, 100 nM) for 1, 2, and 6 hours. Western blot analysis was used to assess the extent of degradation of protein substrates Aiolos, Ikaros, and ZFP91. Compound 1 inhibited Aiolos, Ikaros, and ZFP91 as early as 1 hour with treatments as low as 1 nM. All three proteins showed similar degradation rates, with over 59% of each of the three proteins degraded within 2 hours with 1 nM compound 1. Compound 2 also reduced the expression of Aiolos, Ikaros, and ZFP91, albeit with different kinetics (Figure 16). For example, decreased expression of Aiolos, Ikaros, and ZFP91 was observed at higher concentrations of Compound 2 and with longer exposure times to Compound 2.

[0341] In a second assay, DF15 cell lines expressing ePL-tagged target protein substrates were used as a model system, and protein degradation was monitored at 0.75, 1, 1.5, 3, 4, and 24 hours over a concentration range of 1 pM to 10 μM. Proteins evaluated were Ikaros (FIG. 17), Aiolos, and ZFP91. Both Compound 1 and Compound 2 were able to degrade the substrate proteins at all time points (Table 9). In further assays, additional time points were included to monitor the effect of treatment with Compound 1 and Compound 2. The results showed that Compound 1 and Compound 2 degraded Aiolos, Ikaros, and ZFP91 in a concentration- and time-dependent manner in DF-15 cells expressing Enhanced ProLabel (ePL)-Aiolos, ePL-Ikaros, or ePL-ZFP91 after 1, 2, 6, or 24 hours of exposure, confirming that the cells were more sensitive to Compound 1 compared to Compound 2.

[0342] The depth of substrate protein degradation, as described by the Y constant, was also measured in this assay relative to a negative control luciferase inhibitor (CC1071297, Y-constant = 0). Both enantiomers showed similar Y-constant values ​​(Table 9), which correspond to similar low Y asymptote in the drug response curve (Figure 17).

[0343] Table 9: Comparison of the kinetics of substrate degradation induced by compound 1 and its R-enantiomer in DF15 cells. [Table 10] EC 50 = half maximal effective concentration; ZFP91 = zinc finger protein 91

[0344] Taken together, these results demonstrate that the levels of Ikaros, Aiolos, and ZFP91 can serve as markers of response to both Compound 1 and Compound 2.

[0345] Example 19: Effect of Compound 1 on the maturation of neutrophil precursors The effect of compound 1 on the maturation of bone marrow progenitor cells to neutrophils was examined using bone marrow CD34 + Ex vivo cultures of cells were evaluated and different dose schedules were evaluated to gain insight into the schedule-dependence of these events.

[0346] Myeloid differentiation was induced by adding stem cell factor (SCF), FMS-related tyrosine kinase 3 ligand (FLT3-L) and granulocyte colony-stimulating factor (G-CSF) to the culture medium. Cell differentiation with or without compound 1 was monitored at the indicated time points by flow cytometry to identify five subpopulations: (1) hematopoietic stem cells (HSC, CD34 + / CD33 - / CD11b - );(2) Stage I cells (CD34 + / CD33 + / CD11b - );(3) Stage II cells (CD34 - / CD33 + / CD11b - );(4) Stage III cells (CD34 - / CD33 + / CD11b + ) and (5) stage IV cells (CD34 - / CD33 - / CD11b + ) cells (immature to mature). Differentiation and viability were monitored every 2 or 3 days during the complete assay.

[0347] The effect of different exposure periods of compound 1 (14 or 5 days of treatment) on the survival and maturation of bone marrow progenitors to neutrophils was assessed by flow cytometry at consecutive concentrations of compound 1, 10, and 100 nM for the indicated times up to 21 days (Figure 18; Figure 19). The results showed that late maturation of neutrophil progenitors was blocked by compound 1, with a significant decrease in the number of mature neutrophils at higher concentrations after 14 days (Figure 19A) or 5 days (Figure 19B) of exposure. Maturation arrest appears to occur primarily at the development of stage III neutrophil progenitors, as revealed by the accumulation of cells with stage III cell surface immunophenotype and a decrease in the population of cells with stage IV cell surface immunophenotype (mature neutrophils). However, the survival of neutrophil precursors exposed to compound 1 was not affected (Figure 18).

[0348] Recovery of mature neutrophils following exposure to Compound 1 was also assessed in this system. After 1 week without Compound 1, the percentage of mature neutrophils (stage IV cells) had recovered at least about 50% from its nadir, with a trend toward more rapid and complete recovery at lower concentrations (Figure 20; Table 10).

[0349] Ikaros protein levels were monitored over the course of Compound 1 exposure and recovery. Ikaros levels decreased during Compound 1 exposure and recovered after drug withdrawal in a concentration-dependent manner, with no significant differences noted in relation to the different exposure regimens (Figure 21; Figure 22). Ikaros levels began to return to normal at least 3 days after washout, preceding full recovery of late neutrophil precursor maturation (Figure 23). These results demonstrate that Ikaros degradation of late neutrophil precursors may be an important mediator of neutropenia in Compound 1 recipients. Moreover, the findings suggest that restoration of Ikaros levels precedes recovery of neutrophil progenitor maturation. Thus, Ikaros levels in neutrophils may serve as a marker for response to Compound 1.

[0350] In the in vitro assay system utilized in this study, recovery of mature neutrophil levels to at least 50% of untreated control levels correlates with the absence of clinically significant neutropenia in patients. In this study, after one week without drug, the population of stage IV cells was able to recover in a concentration-dependent manner to levels equivalent to at least 50% of the stage IV cell population in the DMSO control under all test conditions evaluated (Figure 20), even reaching the levels of the DMSO control under some conditions tested.

[0351] Table 10: Amount of time required for stage IV cells to recover to 50% after 14 or 5 consecutive days of exposure to Compound 1 and 1 week of washout after treatment. [Table 11]

[0352] Ikaros protein levels were analyzed by flow cytometry every 2 or 3 days. As shown in Figures 21 and 22, Ikaros protein was degraded under both Compound 1 treatment regimes (14 and 5 days) and its expression recovered in a concentration-dependent manner after washing out of the drug. At day 19, full recovery of Ikaros was observed at all concentrations after 14 days of treatment. Recovery of Ikaros protein expression was slower in cells treated for 5 days than in cells treated for 14 days: at day 19, recovery of Ikaros protein was not complete at any concentration of Compound 1, and at day 21, Ikaros protein levels were fully restored only in cultures exposed to 10 nM of the compound.

[0353] In this study, in cells exposed to compound 1 for 14 days, control levels of Ikaros were restored before neutrophil precursor differentiation was restored, as shown in Figure 23. At concentrations of 10-1000 nM compound 1, restoration of neutrophil precursor maturation occurred more slowly than restoration of Ikaros protein levels.

[0354] Taken together, these results suggest that the levels of Ikaros protein, as well as CD34, in bone marrow cells are increased. + We demonstrate that apoptosis and myeloid differentiation in cells can serve as markers of response to Compound 1.

[0355] The above embodiments are intended to be merely illustrative, and those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific compounds, materials and procedures, all of which are within the scope of the invention and are covered by the appended claims.

[0356] Example 20: Response markers identified from integrated analysis of transcriptome and proteome profiling Biomarkers associated with the cytotoxic effect of Compound 1 treatment in DLBCL, such as signaling and regulatory pathways, were determined by an integrative network using transcriptome and proteome profiling. The method consists of optimizing network flows using knowledge-driven scaffolding networks of protein interactions and regulatory associations similar to those previously described (Basha, Mauer, Simonovsky, Shpringer, & Yeger-Lotem, 2019; Gosline, Spencer, Ursu, & Fraenkel, 2012).

[0357] Briefly, differential expression analysis and differential protein abundance analysis at different time points were integrated using a model framework, which is a tripartite graph that includes a protein-protein interaction (PPi) network layer, a transcriptional regulatory network layer, and pathway definitions compiled from the following sources: Transcriptional Regulatory Network (TRN) -In-house IKZF3 & ZFP91 gene signature and ReMAP (492 TFs) (Cheneby, Gheorghe, Artufel, Mathelier & Ballester, 2018) - ChIP-Seq peaks annotated to transcription start sites (TSS) and filtered for proximity (TSS ±2 kbp) -436 transcription factors and 21,633 genes in the TRN (regulon size ∈ [10,7500]) Protein-protein interaction network (PPiN) -Integrating HINT (Das & Yu, 2012), H-II-14 (Rolland et al., 2014), human soluble protein complexes (Ruepp et al., 2010) & BioPlex (Huttlin et al., 2015) and filtering with reliable experimental evidence -13872 proteins x 144344 edges (after taking directionality into account) Route from MSIgDB-C2 v6.1 -8,904 genes x 1,329 pathways (Subramanian et al., 2005)

[0358] The edge capacity is log 10 The scores were scaled by harmonic mean as standardized log2 fold changes from differential expression and protein abundance analyses normalized by p-value (Xiao et al., 2014). Scores were standardized and scaled between -1 and 1.

[0359] Flux calculations were performed using the maximum flow method as implemented in the graphmaxflow package in Bioconductor. An iterative process was performed to filter out the least relevant interactions while maintaining a significant amount of flux flowing through the network. Edges with flux lower than the 5th quantile were removed in each iteration, and disconnected paths from sources / sinks were removed until an optimization criterion was satisfied.

[0360] First, the sensitivity of cell lines to Compound 1 was measured by flow cytometry in 40 cell line models using DRAQ7 and Annexin V staining to quantify the reduction in proliferation and induction of apoptosis after 5 days of compound treatment. Dose-response curves were generated for each cell line, and the area under the curve (AUC) was calculated. Sensitivity was calculated as (AUC アポトーシス / AUC 生細胞 ) where a smaller percentage indicates resistance and a larger percentage indicates sensitivity. A panel of 11 DLBCL cell lines was found to have various sensitivities to Compound 1 treatment, ranging from resistance to sensitivity.

[0361] Next, 11 DLBCL cell lines covering a broad spectrum of sensitivity to compound 1 were exposed to 0.04 μM compound 1 or DMSO control, and proteomic (TMT-MS) and gene expression (RNA-seq) profiles were obtained in triplicate for a panel. Samples were profiled at five time points after exposure: 6 and 18 hours for proteome; and 12, 24, and 48 hours for transcriptome. A scheme for dynamic integration of proteomic and transcriptomic information is shown in Figure 24. The integrated mechanistic analysis revealed that compound 1 treatment produced Ikaros / Aiolos-driven upregulation of genes associated with interferon signaling (e.g., IL6ST, IFITM3, IFI6, OAS3, interferon α / β signaling), cytokine / chemokine signaling (e.g., IL23A, CCL1), apoptosis (e.g., IL27, TNF, IL10, caspases), cell adhesion (e.g., SELE, SELPLG, TXA2), cell-cell junctions (e.g., CLDN7, CLDN12), G-protein-coupled receptors (e.g., FFAR2), extracellular matrix (e.g., CD209, SERPINA, SERPINB7), and global downregulation of genes related to cell cycle and transcription. An overview of the different pathways for compound 1 sensitivity and the genes associated with the pathways is shown in Figure 25. In particular, several genes were found to be associated with the cytotoxic effect of Compound 1, demonstrating that the activation of these genes can serve as biomarkers for the response of Compound 1 in DLBCL. This list includes interferon and chemokine-related genes (e.g., IL23A, CCL2, IFITM3), cell adhesion genes (e.g., CLDN7), GPCR signaling genes, and apoptosis-related genes (e.g., TNF). For example, by comparing and displaying the fold changes of exemplary genes such as IL23A (Figure 26A), CCL2 (Figure 26B), and SRGAP1 (Figure 26C) across different time points and different cell line models, we have shown that the increased modulation of these markers in cell lines with strong cytotoxic effects was found to be associated with sensitivity to treatment with Compound 1.In addition, comparison of the proteomic results measured at 6 and 18 hours indicated that differences in protein expression may be associated with sensitivity to Compound 1 treatment (Figure 27). For example, protein levels of genes such as IKZF3, IKZF1, ZFP91, ETS1, MNT, MEF2B, SNAPC1, KDM4B, TFAP4, UBTF, BAHD1, MBD4, CBX2, TP63, TLE3, FOXP1, ZBTB11, IRF4, MED26, ATF7, ZNF644, KDM5B, USF2, TCF25, KDM4A, L3MBTL2, SNAPC4, KDM5, EBF1, FOXJ2, NFATC1, ZFP36, HDGF, ELF1, PML, MYBL2, SMAD2, CHD2, STAT1, PAX5, STAT2, PYGO2, IRF9, PCGF2, and ATF3 were found to change in response to treatment with Compound 1, with greater changes observed after 18 hours of treatment compared to 6 hours of treatment.

[0362] Taken together, these results demonstrate that upregulation of genes associated with interferon signaling, cytokine / chemokine signaling, apoptosis, and cell adhesion, and global downregulation of genes associated with cell cycle and transcription, can serve as biomarkers for sensitivity to treatment with Compound 1. In particular, exemplary genes such as IL23A, CCL2, IFITM3, CLDN7, TNF, and SRGAP1 can serve as biomarkers for sensitivity to treatment with Compound 1.

Claims

1. As an active ingredient, a compound of formula (I): 【Chemistry 1】 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof, The active ingredient is used to identify whether a subject with blood cancer may be responsive or to predict the responsiveness of a subject with or suspected of having blood cancer; The identification or prediction may include: (a) obtaining a sample from said subject; (b) measuring the level of a biomarker in the sample in vitro; (c) (i) the level of the biomarker in the sample is detectable; or (ii) diagnosing the subject as likely to be responsive to the active ingredient if the level of the biomarker in the sample is at an altered level compared to the level of a reference biomarker.

2. For the preparation of a medicament for selectively treating blood cancer, 【Chemistry 2】 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, as an active ingredient, wherein the selective treatment (a) obtaining a sample from a subject with a blood cancer; (b) measuring the level of a biomarker in the sample in vitro; (c) (i) the level of the biomarker in the sample is detectable; or (ii) diagnosing the subject as likely to respond to the active ingredient if the level of the biomarker is at an altered level compared to a reference level of the biomarker; and (d) A use comprising administering a therapeutically effective amount of an active ingredient to a subject diagnosed as likely to be responsive to said active ingredient.

3. Biomarkers (a) diagnosing the subject as likely to respond to the pharmaceutical composition if cereblon (CRBN) is detectable or higher than a reference level in the sample; or (b) Ikaros, Aiolos, ZFP91, or a combination thereof; (i) diagnosing that the subject is likely to respond to the active ingredient if the level of the biomarker in the sample is lower than a reference level; or (ii) The pharmaceutical composition according to claim 1, wherein when the levels of both Ikaros and Aiolos are lower than their respective reference levels, the patient is diagnosed as likely to be responsive to the active ingredient.

4. Biomarkers (a) diagnosing the subject as likely to respond to the pharmaceutical composition if cereblon (CRBN) is detectable or higher than a reference level in the sample; or (b) Ikaros, Aiolos, ZFP91, or a combination thereof; (i) diagnosing that the subject is likely to respond to the active ingredient if the level of the biomarker in the sample is lower than a reference level; or (ii) The use according to claim 2, wherein a patient is diagnosed as likely to respond to the active ingredient when the levels of both Ikaros and Aiolos are lower than their respective reference levels.

5. A compound of formula (I) as an active ingredient: 【Chemistry 3】 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof, The active ingredient is used to identify whether a subject with blood cancer may be responsive or to predict the responsiveness of a subject with or suspected of having blood cancer; The identification or prediction may include: (a) obtaining a sample from said subject; (b) adding said therapeutic compound to said sample ex vivo; (c) measuring the level of a biomarker in the sample; and (d) diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is at an altered level compared to the level of a reference biomarker.

6. A compound of formula (I) as an active ingredient: 【Chemistry 4】 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof, The active ingredient is used to identify whether a subject with blood cancer may be responsive or to predict the responsiveness of a subject with or suspected of having blood cancer; The identification or prediction may include: (a) obtaining a sample from a subject administered a therapeutic compound; (b) measuring the level of the biomarker in the sample in vitro; and (c) diagnosing the subject as likely to be responsive to the therapeutic compound if the level of the biomarker in the sample is at an altered level compared to the level of a reference biomarker.

7. As an active ingredient, a compound of formula (I): 【Chemistry 5】 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof, monitoring the efficacy of the active ingredient in treating a blood cancer in a subject; The monitoring is (a) obtaining a sample from a subject administered the pharmaceutical composition; (b) measuring the level of a biomarker in the sample in vitro; and (c) comparing the level of the biomarker in the sample with a reference level of the biomarker, wherein an altered level of the biomarker indicates efficacy of the active ingredient in treating hematological cancer in the subject.

8. As an active ingredient, a compound of formula (I): 【Chemistry 6】 or an enantiomer, mixture of enantiomers, tautomer, isotopologue, or pharma- ceutical acceptable salt thereof, adjusting the dosage or frequency of administration of the active ingredient to treat a subject with blood cancer; The adjustment is (a) obtaining one or more samples at different time points from a subject administered a dose of the active ingredient; (b) monitoring ex vivo the levels of the biomarkers in one or more samples; and (c) adjusting a dosage for subsequent administration of the active ingredient to the subject based on the altered level of the biomarker in the reference sample.

9. 9. The pharmaceutical composition of claim 7 or 8, further comprising administering a therapeutically effective amount of the active ingredient to a subject diagnosed as likely to be responsive to the active ingredient.

10. (a) the altered level of the biomarker in the sample is higher than the reference level of the biomarker; (b) the altered level of the biomarker in the sample is lower than the reference level of the biomarker; (c) an increase in the level of the biomarker, relative to the reference biomarker level, indicates efficacy of the active ingredient in treating a hematological cancer in the subject. (d) a decrease in the level of the biomarker, relative to the reference biomarker level, indicates efficacy of the active ingredient in treating hematological cancer in the subject. (e) the reference biomarker level is the level of the biomarker in a reference sample obtained from the subject prior to administration of the active ingredient to the subject, the reference sample being from the same source as the sample; (f) the reference biomarker level is the level of the biomarker in a reference sample taken from a healthy subject not suffering from a hematological cancer, the reference sample being from the same source as the sample; or (g) The pharmaceutical composition of claim 5 or 6, wherein the reference biomarker level is a predetermined biomarker level.

11. The pharmaceutical composition of claim 5 or 6, wherein the biomarker comprises a marker of apoptosis, and an alteration in the level of the biomarker indicates induction of apoptosis.

12. The biomarkers are (a) selected from the group consisting of cleaved caspase 3, cleaved caspase 7, cleaved poly(ADP-ribose) polymerase (PARP), BCL2, survivin, phosphatidylserine (PS) and DNA, Bcl-2-like protein 11 (BIM), tumor necrosis factor (TNF), interleukin-10 (IL-10), and interleukin-27 (IL27), or a combination thereof; (b) selected from the group consisting of annexin-V, 7-amino-actinomycin D (7-AAD), and deep red anthraquinone 7 (DRAQ7), or a combination thereof; or 12. The pharmaceutical composition of claim 11, wherein (c) the antibody is selected from the group consisting of IL-8, IL-1a, sPGE2, sTNFα, sIgG, sIL-17A, sIL-17F, sIL-2, sIL-6, collagen-I and -III, PAI-1, CD69, and sIL-10, or a combination thereof.

13. (a) the level of the biomarker in the sample is higher than a reference level of the biomarker, or The pharmaceutical composition of claim 12, wherein (b) the biomarker in the sample is lower than a reference level of the biomarker.

14. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is associated with interferon signaling.

15. 15. The pharmaceutical composition of claim 14, wherein the biomarker comprises interleukin-6 signal transducer (IL6ST), interferon-induced transmembrane protein 3 (IFITM3), interferon alpha-inducible protein 6 (IFI6), 2'-5'-oligoadenylate synthetase 3 (OAS3), interferon alpha (IFNα), interferon beta (IFNβ), or a combination thereof.

16. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is associated with cytokine / chemokine signaling.

17. 17. The pharmaceutical composition of claim 16, wherein the biomarker comprises interleukin-23 subunit alpha (IL23A), C-C motif chemokine 1 (CCL1), or a combination thereof.

18. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is associated with cell adhesion.

19. 19. The pharmaceutical composition of claim 18, wherein the biomarkers include E-selectin (SELE), P-selectin glycoprotein ligand 1 (SELPLG), thromboxane A2 (TXA2), or a combination thereof.

20. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is associated with cell-cell junctions.

21. 21. The pharmaceutical composition of claim 20, wherein the biomarker comprises claudin 7 (CLDN7), claudin 12 (CLDN12), or a combination thereof.

22. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is a G-protein coupled receptor.

23. 23. The pharmaceutical composition of claim 22, wherein the biomarker comprises free fatty acid receptor 2 (FFAR2).

24. The pharmaceutical composition of claim 5 or 6, wherein the biomarker is associated with the extracellular matrix.

25. 25. The pharmaceutical composition of claim 24, wherein the biomarkers include CD209, SERPINA, SERPINB7, or a combination thereof.

26. (a) the biomarker is associated with the cell cycle; (b) the biomarker is associated with a transcript; (c) the biomarker is selected from the group consisting of cyclin-dependent kinase inhibitor 1 (p21), or The pharmaceutical composition of claim 5 or 6, wherein (d) the biomarkers include PD1 and LAG3.

27. Biomarkers include Aiolos (IKZF3), Ikaros (IKZF1), E3 ubiquitin-protein ligase ZFP91 (ZFP91), protein C-ets-1 (ETS1), and MAX binding protein MNT (MNT), myocyte-specific enhancer factor 2B (MEF2B), snRNA activator protein complex subunit 1 (SNAPC1), lysine-specific demethylase 4B (KDM4B), transcription factor AP-4 (TFAP4), nuclear pole transcription factor 1 (UBTF), bromo-adjacent homology domain-containing 1 protein (BAHD1), methyl-CpG-binding domain protein 4 (MBD4), chromobox protein homolog 2 (CBX2), tumor protein 63 (TP63), transducin-like enhancer protein 3 (TLE3), forkhead box protein P1 (FOXP1), zinc finger and BTB domain-containing protein 11 (ZBTB11), interferon regulatory factor 4 (IRF4), mediator of RNA polymerase II transcription subunit 26 (MED26), cyclic A MP-dependent transcription factor ATF-7 (ATF7), zinc finger protein 644 (ZNF644), lysine-specific demethylase 5B (KDM5B), upstream stimulatory factor 2 (USF2), transcription factor 25 (TCF25), lysine-specific demethylase 4A (KDM4A), lethal (3) malignant brain tumor-like protein 2 (L3MBTL2), mRNA-activator protein complex subunit 4 (SNAPC4), lysine-specific demethylase 5 (KDM5), transcription factor COE1 (EBF1), forkhead box protein J2 (FOXJ2), nuclear factor of activated T cells cytoplasm 1 (NFATC1), mRNA downregulation activator protein ZFP36 (ZFP36), hepatoma-inducible growth factor (HDGF), ETS-related transcription factor Elf-1 (ELF1), promyelocytic leukemia protein (PML), Myb-related protein B7. The pharmaceutical composition of claim 5 or 6, comprising one or more proteins selected from the group consisting of MYBL2, mother against decapentaplegic homology 2 (SMAD2), chromodomain-helicase-DNA-binding protein 2 (CHD2), signal transducer and activator of transcription 1 (STAT1), paired-box protein Pax5 (PAX5), signal transducer and activator of transcription 2 (STAT2), pygopathic homolog 2 (PYGO2), interferon regulatory factor 9 (IRF9), polycomb group RING finger protein 2 (PCGF2), and cyclic AMP-dependent transcription factor ATF-3 (ATF3).

28. 7. The pharmaceutical composition of claim 5 or 6, wherein the biomarker comprises one or more genes selected from the group consisting of interleukin-23 subunit alpha (IL23A), C-C motif chemokine 2 (CCL2), and SLIT-ROBO Rho GTPase-activating protein 1 (SRGAP1).

29. 7. The pharmaceutical composition of claim 5 or 6, wherein the biomarker comprises a CRBN binding protein or a transcriptional target of a CRBN binding protein.

30. (a) the CRBN binding protein is selected from the group consisting of Ikaros, Aiolos, and ZFP91, or 30. The pharmaceutical composition of claim 29, wherein (b) the transcriptional target of the CRBN binding protein is selected from the group consisting of BCL6, c-MYC, and IRF4.

31. 30. The pharmaceutical composition of claim 29, wherein the biomarker comprises an interferon-inducible gene.

32. 32. The pharmaceutical composition of claim 31, wherein the biomarker is selected from the group consisting of interferon-regulated 7 (IRF7), interferon-induced protein with tetratricopeptide repeat unit 3 (IFIT3), DEAD box protein 58 (DDX58), or a combination thereof.

33. The pharmaceutical composition of claim 5 or 6, wherein the biomarker comprises a marker of T cell activation.

34. 34. The pharmaceutical composition of claim 33, wherein the marker of T cell activation comprises a T cell activation-associated cytokine.

35. The pharmaceutical composition of claim 34, wherein the T cell activation-associated cytokine comprises interleukin 2 (IL-2).

36. The biomarker comprises an effector cytokine or an effector chemokine; The pharmaceutical composition according to claim 5 or 6.

37. 37. The pharmaceutical composition of claim 36, wherein the biomarker is selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and combinations thereof.

38. The pharmaceutical composition of any one of claims 14 to 34, wherein the biomarker in the sample is higher than a reference level of the biomarker.

39. The pharmaceutical composition of any one of claims 14 to 34, wherein the biomarker in the sample is lower than a reference level of the biomarker.

40. The pharmaceutical composition of any one of claims 5, 6, 10 to 39, wherein the biomarker is expressed in leukocytes.

41. 41. The pharmaceutical composition of claim 40, wherein the white blood cells comprise lymphoid cells.

42. 42. The pharmaceutical composition of claim 41, wherein the lymphoid cells comprise T cells.

43. For the manufacture of a medicament for treating blood cancer, 【Chemistry 7】 or an enantiomer, a mixture of enantiomers, a tautomer, an isotopologue, or a pharma- ceutically acceptable salt thereof, as an active ingredient, wherein the treatment comprises (a) obtaining a first sample from a subject with hematological cancer; (b) measuring in vitro the level of a biomarker in a first sample; (c) administering to the subject a therapeutically effective amount of the active ingredient; (d) obtaining at least one additional sample from the subject after administration of the treatment; and (e) measuring the level of the biomarker in at least one additional sample; If the level of the biomarker in the at least one further sample is at or near the level of the biomarker in the first sample, then administering to the subject another therapeutically effective amount of the active ingredient. 。

44. 44. The use of claim 43, wherein the biomarker comprises Ikaros.

45. 45. The use of claim 44, wherein the biomarker is expressed in leukocytes.

46. 46. ​​The use of claim 45, wherein the white blood cells comprise bone marrow cells.

47. 47. The use of claim 46, wherein the bone marrow cells comprise neutrophils.

48. Biomarker is phenotype CD11b ＋ , CD34 － , and CD33 － 44. The use of claim 43, comprising neutrophils which are

49. The compound is (a) (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof; (b) (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or a tautomer, isotopologue, or a pharma- ceutically acceptable salt thereof; (c) a mixture of (S)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione with (R)-2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dione, or with a tautomer, isotopologue, or pharma- ceutically acceptable salt thereof. The pharmaceutical composition according to any one of claims 5, 6, 10 to 42, comprising:

50. 50. The pharmaceutical composition of any one of claims 5, 6, 10-42, 49, further comprising administering a therapeutically effective amount of a second active agent or a supportive care treatment.

51. The second active agent is an HDAC inhibitor (e.g., panobinostat, romidepsin, vorinostat, or citalinostat), a BCL2 inhibitor (e.g., venetoclax), a BTK inhibitor (e.g., ibrutinib or acalabrutinib), an mTOR inhibitor (e.g., everolimus), a PI3K inhibitor (e.g., idelalisib), a PKCβ inhibitor (e.g., enzastaurin), a SYK inhibitor (e.g., fostamatinib), a JAK2 inhibitor (e.g., fedratinib, pacritinib, ruxolitinib, baricitinib, gandotinib, lestaurtinib, or momelotinib), an Aurora A kinase inhibitor (e.g., alisertib), an EZH2 inhibitor (e.g., tazemetostat, GSK126, CPI-1205, 3-deazaneplanocin A, 51. The pharmaceutical composition of claim 50, wherein the therapeutic agent is selected from the group consisting of a medicament for treating rheumatoid arthritis (e.g., ribavirin, EPZ005687, EI1, UNC1999, or sinefungin), a BET inhibitor (e.g., virabresib or 4-[2-(cyclopropylmethoxy)-5-(methanesulfonyl)phenyl]-2-methylisoquinolin-1(2H)-one), a hypomethylating agent (e.g., 5-azacytidine or decitabine), a chemotherapeutic agent (e.g., bendamustine, doxorubicin, etoposide, methotrexate, cytarabine, vincristine, ifosfamide, melphalan, dexamethasone, or oxaliplatin), an anti-CD20 monoclonal antibody (e.g., rituximab or obinutuzumab), an epigenetic compound, or a combination thereof.

52. The second active agent (a) rituximab, (b) obinutuzumab 52. The pharmaceutical composition of claim 51 , comprising:

53. The pharmaceutical composition of any one of claims 5, 6, 10-42, 49-52, wherein the hematological cancer affects hematopoietic or lymphoid tissue.

54. The pharmaceutical composition of any one of claims 5, 6, 10-42, 49-53, wherein the blood cancer comprises non-Hodgkin's lymphoma.

55. 55. The pharmaceutical composition of claim 54, wherein the non-Hodgkin's lymphoma comprises diffuse large B-cell lymphoma (DLBCL).

56. 56. The pharmaceutical composition of claim 55, wherein the DLBCL is relapsed, refractory or resistant to conventional therapy.

57. 54. The pharmaceutical composition of claim 53, wherein the hematological cancer comprises chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL).

58. 58. The pharmaceutical composition of claim 57, wherein the CLL / SLL is relapsed, refractory or resistant to conventional therapy.

59. The pharmaceutical composition of any one of claims 5, 6, 10-42, 49-58, wherein the sample comprises blood cancer cells.

60. Measuring the levels of biomarkers (a) the protein level of a biomarker; (b) the level of mRNA of a biomarker; or (c) Biomarker cDNA levels The pharmaceutical composition according to any one of claims 5, 6, 10 to 42, and 49 to 59, comprising measuring

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