Methods of treating chronic lymphocytic leukemia

Abstract

There is provided a method of treating chronic lymphocytic leukemia (CLL) comprising the administration of a therapeutically effective amount of a FLT3 inhibitor and, optionally, one or more additional agents which are suitable for the treatment of CLL to a patient in need thereof.

Description

[0001] METHODS OF TREATING CHRONIC LYMPHOCYTIC LEUKEMIA

[0002] FIELD

[0003] There are provided methods of treatment of chronic lymphocytic leukemia (CLL). In particular, the methods comprise administration of a feline McDonough sarcoma like receptor tyrosine kinase 3 (FLT3) inhibitor.

[0004] BACKGROUND

[0005] CLL is a type of cancer in which too many blood stem cells become abnormal lymphocytes. The abnormal lymphocytes may also be called leukemia cells. These leukemia cells are not able to fight infection very7well. Also, as the number of leukemia cells increases in the blood and bone marrow, there is less room for healthy white blood cells, red blood cells, and platelets. This may lead to infection, anemia, and easy bleeding.

[0006] It is known that CLL is dependent on microenvironmental cues for survival and expansion. CLL cells receive survival signals via accessory7cells like mesenchymal stroma cells, nurse-like cells, and T-cells residing in the red bone marrow microenvironment. Interaction with microenvironmental stimuli results in activation of downstream signaling pathways that promote CLL survival and increases resistance to drug-induced apoptosis. CLL cells typically undergo spontaneous apoptosis within 24 hours when cultured in vitro in simple media supplemented only with fetal bovine serum. In vitro studies have demonstrated that bone marrow derived stromal cells protect CLL cells from apoptosis and support their survival. It is notable that others have found that stromal cell support required cell-cell contact, as they found separation across micropore filters reduced the protective effect.

[0007] Studies have suggested that CC motif chemokine ligand 2 (CCL2) may play an important role in the survival of CLL cells which are supported by stromal cell media (Parvin et al., Blood Cancer Discov.. 2022, 3. 5 supplement, A39). However, these studies also identified a subset of CLL cells that were resilient to cell death even in the absence of stromal support in ex-vivo culture. Unlike CLL cells which were dependent on stromal support, anti- CCL2 antibody (either alone, or in combination with venetoclax) showed no effect on the survival of this subset of media independent CLL cells.

[0008] FLT3 inhibitors are known for treating AML (see Hassanein et al., Clin. Lymphoma Myeloma Leuk. , 2016, 16, 10, 543-549 and Zhao et al., Blood Rev., 2022, Mar: 52: 100905, doi: 10.1016 / j.blre.202L 100905. Epub 2021 Nov 3). The first-generation FLT3 inhibitors were developed several years ago and include midostaurin, lestaurtinib, sunitinib, and sorafenib. They are relatively nonspecific for FLT3, with other potential targets that include platelet- derived growth factor receptor, vascular endothelial growth factor receptor, the KIT tyrosineprotein kinase (also known as the stem cell growth factor receptor (SCFR)), and Janus kinase 2. The second-generation inhibitors, including quizartinib. crenolanib, PLX3397, and ASP2215 (gilteritinib), are more potent and selective than the first-generation inhibitors. The greater potency and selectivity promises greater efficacy in FLT3-mutated acute myelogenous leukemia (AML) (particularly in patients with a greater allele burden) and less toxicity. A number of receptor tyrosine kinase inhibitors are being studied across virtually all disease settings, including frontline, relapsed and refractory, and maintenance, mainly in patients with FLT3-mutated AML.

[0009] Some studies have also been performed wherein compounds which are capable of inhibiting FLT3 have shown effects in the treatment of CLL. Sorafenib has been shown to be capable of targeting a variety of human lymphomas representative of different phenotypes by inhibiting tumor angiogenesis and directly affecting tumor cell survival (Carlo-Stella et al., Blood, 2008, 112 (11), 2605). It has also been shown to suppress angiogenic pathways in CLL by inhibiting secretion of IL-8 and expression of neuropilin- 1 (Reganti et al, Blood, 2007, 110 (11), 4726); and to induce apoptosis in CLL samples, including those bearing adverse cytogenetic alterations (Lopez-Guerra et al., 2010, Eur. J. Cancer Supplement, vol. 8, no. 7, p.57-58). Luxeptinib (CG-806), a pan-FLT3 / pan-BTK inhibitor, has been shown to exhibit broad signalling inhibition in CLL cells (Kim et al., Blood, 2019, 134, Supplement 1, 3051); to have anti-tumour activity’ in CLL (Samaniego et el.. Blood, 2022, 140, Supplement 1, 6498- 6500); and to exhibit growth inhibition as a single agent and in combination with a BET bromodomain inhibitor or a BCL2 Inhibitor in primary AML and CLL patient samples. Imidacrine (Symadex®, C-1311) has been shown to reduce the viability of B-CLL cells ex vivo (W02008 / 016660). Quizartinib, in combination with ibrutinib, and sorafenib, in combination with JQ-1, have also been shown to be effective against CLL patient samples (WO2018 / 081830). 1 -{3-fluoro-4-[7-(5-methyl-lH-imidazol-2-yl)-l -oxo-2,3-dihydro-lH- isoindol-4-yl]-phenyl}-3-(2,4,6-trifluoro-phenyl)-urea has been shown to bind to FLT3 and demonstrates cytotoxicity versus various cell lines including one of CLL type (WO2018 / 156578). However, in these disclosures, the drug involved exhibits activity against targets other than FLT3 and it is not clear which of these targets is really important. Furthermore, none of them disclose specific targeting of CLL cells which are unmutated and / or resilient to cell death even in the absence of stromal support in ex-vivo culture. Despite the broad incorporation of highly effective targeted therapies such as Bruton's tyrosine kinase (BTK) inhibitors, B cell receptor (BCR) pathway inhibitors, and B-cell lymphoma 2 (BCL2) antagonists, such as venetoclax, for the treatment of CLL, the disease remains incurable. Furthermore, venetoclax resistance has recently emerged. Identifying alternative and / or complementary therapeutic strategies is therefore necessary.

[0010] SUMMARY

[0011] Such altemative / complementary methods are provided herein. More specifically, following the discovery that CLL cells can be classified based upon their ability to survive in vitro in the presence or absence of conditioned media (CM), CM-dependent (CMD) CLL cells or CM-independent (CMI) CLL cells, respectively, it was discovered that the resistant CMI- CLL showed high expression of the type III receptor tyrosine kinase protein FLT3 and that treatment with a FLT3 inhibitor resulted in a decrease in cell viability in this particular CLL subtype. This discovery has opened up new therapeutic opportunities for the treatment of CLL, in particular when used in conjunction with diagnostics for identifying the CLL subtype of the patient, i.e. CMD or CMI.

[0012] In a first aspect, there is provided a method of treating CLL comprising the administration of a therapeutically effective amount of a FLT3 inhibitor to a patient in need thereof. Also provided is a FLT3 inhibitor for use in a method of treating CLL comprising the administration of said FLT3 inhibitor to a patient in need thereof. Also provided is the use of a FLT3 inhibitor in the manufacture of a medicament for the treatment of CLL in a patient in need thereof. In preferred embodiments, the patient has been previously identified as suffering from CLL which is CM-independent or, more specifically, CCL2 independent. In further preferred embodiments, the patient has been previously identified as suffering from CLL which is resistant to treatment with a BCL2 antagonist. In a preferred embodiment, the FLT3 inhibitor is the sole active ingredient used in the method of treatment; that is to say, the FLT3 inhibitor is effective as monotherapy.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A illustrates the establishment of conditioned media and its use for culturing CLL.

[0015] Figure IB illustrates relative CLL cell viability when grown in presence of either CM, BM or NKtert Cells for 4 days, n=5. Figure 1C illustrates Annexin V staining of CLL cells after two days in culture with CM or BM.

[0016] Figure 2A illustrates viability of PBMCs isolated from 23 CLL patients in BM.

[0017] Figure 2B illustrates cell death in PBMC samples whose viability is >75% (CMI) and in PMBC samples whose viability is <75% (CMD).

[0018] Figure 2C illustrates cell viability of PBMCs from both groups (CMD and CMI) grown in CM and treated with increasing doses of venetoclax (ABT-199)

[0019] Figure 2D illustrates IC50 values of venetoclax (ABT- 199) segregate based on CM dependence.

[0020] Figure 2E, Upper illustrates Western blot analysis of whole cell extracts using the MCL1 antibody from both CLL groups.

[0021] Figure 2E, Lower illustrates densitometry analysis for images shown in upper panel.

[0022] Figure 2F, Upper illustrates a heatmap of BH3 profile performed on freshly isolated CLL-PBMCs from each group using BIM and BAD peptides.

[0023] Figure 2F, Lower shows the difference in cytochrome C release in response to BIM and BAD peptides between both groups.

[0024] Figure 2G. Illustrates cell viability of PBMCs in BM according to IgVH status; the table shows the odds ratio is ~ 17: 1 between IgHV-U given CMI status as opposed to IgHV- M.

[0025] Figure 3A compares CLL patients' PBMCs from both groups for either CD3+ or CD14+.

[0026] Figure 3B compares cell viability for CD 19+ cells isolated from CLL PBMCs from both groups.

[0027] Figure 3C, Upper illustrates hierarchical clustering of gene expression data of CM dependent vs. CM independent CLL; Lower illustrates PCA analysis of different CM dependent and independent samples.

[0028] Figure 3D is a heatmap showing differentially expressed genes between CM dependent vs. CM independent CLL.

[0029] Figure 3E illustrates signaling pathways enriched for the differentially expressed genes.

[0030] Figure 3F illustrates relative expression of genes in RAS signaling pathway and Focal adhesion.

[0031] Figure 4A, Left illustrates FLT3 gene expression assessed by RT-qPCR; Right illustrates surface expression of FLT3 in PBMCs from each CLL group Figure 4B illustrates cell viability of primary CLL samples from each group following 72 hours of FLT3 inhibitor treatment.

[0032] Figure 5A illustrates that CLL samples undergo little spontaneous cell death when cultured in BM with CM supplementation.

[0033] Figure 5B is a heatmap of baseline profile performed on freshly isolated CLL-PBMCs.

[0034] Figure 5C compares CLL survival when grown in CM derived from HS-5 stroma cell line with CLL cells that are cocultured directly with HS-5 cells.

[0035] Figure 5D illustrates CMI expressed higher IL8 mRNA levels compared to CMD.

[0036] Figure 5E illustrates CMI samples showed increased sensitivity to FLT3 inhibitor gilteritinib compared to CMD samples.

[0037] Figure 5F illustrates CMI samples showed increased sensitivity to FLT3 inhibitor quizartinib compared to CMD samples. gd

[0038] DETAILED DESCRIPTION

[0039] Definitions

[0040] A “BCL2 antagonist'’ is a substance which binds to BCL2 and prevents it from carrying out its function of inhibiting apoptosis. Suitable BCL2 antagonists are known in the art or may be identified by measuring their ability7to bind BCL2 and / or kill cells that are dependent on BCL2 for survival. Preferred BCL2 antagonists exhibit a dissociation constant (Kd) of less than 100 nM in a biochemical binding assay with the BCL2 protein, for example in commercially available assays such as BCL2scan from Eurofms / DiscoverX

[0041] TR-FRET Assay Kit from Bioscience (https: / / bpsbioscience.com / bcl-2-tr-fret-assay-kit- 50222). Suitable BCL2 antagonists include BCL-2 degraders which also bind BCL2, but antagonize the function of BCL2 by facilitating its degradation, thereby lowering levels of the BCL2 protein in the cell of interest. Suitable BCL2 antagonists also include antisense oligonucleotides that inhibit expression of BCL2.

[0042] A “FLT3 inhibitor” is a substance which binds to the FLT3 protein (preferably to the catalytic domain thereof) and thereby inhibits the kinase activity of the FLT3 protein. Suitable FLT3 inhibitors are known in the art or may be identified by their ability to bind to the FLT3 protein in a suitable binding assay and / or inhibit FLT3 dependent phosphorylation of FLT targets (including itself via autophosphorylation, and the protein BAD) in a suitable functional assay. Preferred FLT3 inhibitors exhibit a dissociation constant (Kd) of less than 100 nM in a biochemical binding assay with the FLT3 protein, for example in the competition binding assay used in Chao et al., J.Med.Chem., 2009. Vol. 52, No.23 and further described in Fabian et al., Nat. Biotechnol., 2005, Mar;23(3):329-36. Additionally, or alternatively, preferred FLT3 inhibitors selectivity inhibit FLT3 over other biological targets which are known to play a role in CLL. in particular over other kinases which are known to play a role in CLL. In other words, the FLT3 inhibitor binds to the FLT3 protein and inhibits its kinase activity at a significantly lower concentration than that at which it binds (if it binds at all) to other biological targets which are known to play a role in CLL, in particular over other kinases which are known to play a role in CLL. Especially preferred FLT3 inhibitors exhibit at least 10-fold, more preferably at least 100-fold, selectivity for inhibition of FLT3 over other biological targets which are known to play a role in CLL, in particular over other kinases which are known to play a role in CLL.

[0043] The term “conditioned media dependent CLL cells” or “CMD-CLL” means CLL cells which exhibit prolonged survival ex vivo in the presence of media conditioned by bone marrow' stromal cells compared to their survival ex vivo in unconditioned media. More specifically, CMD-CLL are defined as those w'here less than 75% of the CLL population survives a 24 hour incubation in culture media lacking media conditioned by bone marrow stromal cells.

[0044] The term “conditioned media independent CLL cells” or “CMI-CLL” means CLL cells which do not exhibit prolonged survival ex vivo in the presence of media conditioned by bone marrow stromal cells compared to their survival ex vivo in unconditioned media. More specifically, CMI-CLL are defined as those where greater than or equal to 75% of the CLL population survives a 24 hour incubation in culture media lacking media conditioned by bone marrow' stromal cells.

[0045] The term “combined administration” means administration of the two components of the combination in a manner that results in them exerting their desired pharmacodynamic effects at the same time within the patient’s body. Thus, combined administration is not limited to simultaneous administration of the tw'O components, nor administration via the same route. It encompasses separate, sequential and simultaneous administration via the same or different routes and in the same or different medicaments provided that it results in them exerting their desired pharmacodynamic effects at the same time within the patient’s body.

[0046] Treatment of CLL

[0047] The inventors chose to investigate whether the soluble factors present in media conditioned by a bone marrow -derived stromal cell line could impact CLL cell survival in short term ex-vivo culture. In particular, the inventors chose to investigate whether media conditioned by supporting cells (conditioned media, CM) could replace the more complicated existing co-culture systems. It was found that CM support depended on the presence of a finite number of cytokines, mostly on CC motif chemokine ligand 2 (CCL2).

[0048] Two groups of primary CLL cells have been identified: one group relying on CM for their survival in vitro (hereafter referred to as “CM-dependent CLL cells”, or '‘CMD-CLL”) and another group with resistance to apoptosis even in the absence of CM support (hereafter referred to as “CM-independent cells”, or “CMI-CLL”). These two distinct groups were distinguishable by a simple 24-hour functional assay. Furthermore, combined treatment with anti-CCL2 antibody and the BCL2 antagonist venetoclax has been shown to markedly reduce CMD-CLL survival more effectively than monotherapy.

[0049] Further investigations resulted in the remarkable discovery that the resistant CMI-CLL showed high expression of the type III receptor tyrosine kinase protein FLT3 and that treatment with a FLT3 inhibitor resulted in a decrease in cell viability in this particular CLL subtype. Although FLT3 (which is expressed on the surface of many hematopoietic stem cells and is important for their normal development) is a known proto-oncogene, up to now mutations in FLT3 have only been associated with myelogenous leukemia, in particular, acute myelogenous leukemia (AML) in which it is characterized as the most frequent genetic alteration, detected in 30% of AML patients and confers poor outcome. Indeed, FLT3 inhibitors, such as gilteritinib and quizartinib have been used for the treatment of patients with AML. However, it was not previously known that FLT3 played any role in lymphocytic leukemias, such as CLL.

[0050] There is provided a method of treating CLL comprising the administration of a therapeutically effective amount of a FLT3 inhibitor to a patient in need thereof. Also provided is a FLT3 inhibitor for use in a method of treating CLL comprising the administration of said FLT3 inhibitor to a patient in need thereof. Also provided is the use of a FLT3 inhibitor in the manufacture of a medicament for the treatment of CLL in a patient in need thereof.

[0051] In a preferred embodiment, the FLT3 inhibitor is the sole active ingredient used in the method of treatment; that is to say, the FLT3 inhibitor is effective as monotherapy. However, there is also provided a method of treating CLL comprising the combined administration of therapeutically effective amounts of a FLT3 inhibitor and one or more additional agents w hich are suitable for the treatment of CLL to a patient in need thereof. In this context, the ■‘therapeutically effective amounts” of the FLT3 inhibitor and one or more additional agents encompass amounts of each when used for combined administration. This means that the amounts may be lower than those required when such substances are used as monotherapy. Also provided is a FLT3 inhibitor and one or more additional agents which are suitable for the treatment of CLL for use in a method of treating CLL comprising the administration of said FLT3 inhibitor and said one or more additional agents which are suitable for the treatment of CLL to a patient in need thereof. Also provided is the use of a FLT3 inhibitor and one or more additional agents which are suitable for the treatment of CLL in the manufacture of a medicament for the treatment of CLL in a patient in need thereof.

[0052] In preferred embodiments, the patient has been previously identified as suffering from CLL which is CM-independent or, more specifically, CCL2 independent. CM-independent cases of CLL can be identified by observing the survival of CLL cells in ex vivo culture (see Example 1 below). When greater than or equal to 75% of the CLL population survives a 24 hour incubation in culture media lacking media conditioned by bone marrow stromal cells, the case of CLL can be considered CM-independent. Alternatively, CM-independent cases of CLL can be identified by analyzing IGHV mutational status (see Example 2 below). CMI CLL have unmutated-IGHV. Alternatively. CM-independent cases of CLL can be identified by analyzing IL8, CCL2 and IL6 mRNA levels (see Example 3 below). CMI CLL express high levels of IL8 compared to CCL2 and IL6.

[0053] In further preferred embodiments, the patient has been previously identified as suffering from CLL which is resistant to treatment with a BCL2 antagonist.

[0054] Suitable FLT3 inhibitors of use include gilteritinib, quizartinib, midostaurin, luxeptinib (CG-806), sorafenib, tandutinib, lestaurtinib, imidacrine (C-1311 (Symadex®)) and crenolanib. Preferably, the FLT3 inhibitor is gilteritinib or quizartinib.

[0055] Additional agents which are suitable for the treatment of CLL include, but are not limited to, BCL-2 inhibitors (such as venetoclax, BGB-11417. ZN-D5, APG-2575, TQB3909, LOXO-338, ABBV-453, LP-108 and BP1002), PI3k inhibitors (such as idelalisib, copanlisib, umbralisib and duvelisib), BTK inhibitors (such as ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib and orelabrutinib), CD20 antibodies (such as obinutuzumab, rituximab, ofatumumab. ocrelizumab and veltuzumab). corticosteroids (such as prednisone, dexamethasone and methylprednisolone) and chemotherapeutic agents (such as cyclophosphamide and fludarabine).

[0056] EXAMPLES

[0057] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Those of ordinary skill in the art may be aware of materials and methods similar or equivalent to those described below and any of these can be used to practice or test the provided methods and compositions. Figure 1 illustrates the establishment of conditioned media for CLL. In Figure 1A, stromal NKTert cell line is cultured in complete media containing RPMI supplemented with 10% FBS and penicillin-streptomycin (basal media, BM). Once the cells reach around 90% confluency. the supernatant is collected, filtered and stored at -80°C. Mononuclear cells, isolated from peripheral blood of CLL patient samples, is cultured in presence of this conditioned media (100%). >90% cells are CD19+ CD5+. Figure IB illustrates relative CLL cell viability when grown in presence of either CM, BM or NKtert Cells for 4 days, n=5. Figure 1C illustrates Annexin V staining of CLL cells after two days in culture with CM or BM. n=4, Error bars represent SEM, P values are calculated using paired t-test, P value < 0.05 indicates statistical significance.

[0058] Figure 2 illustrates that a subgroup of CLL does not undergo spontaneous apoptosis in absence of stroma support. Figure 2A. PBMCs are isolated from 23 CLL patient samples and plated in 384-well plates in BM. After 24 hours, cell viability is measured using Cell Titer- Glo. The horizontal line represents 75% cell viability. Percent Cell viability is calculated relative to Day 0 (100%). Figure 2B, cell death is measured in seven PBMC samples whose viability is >75% (CMI) and in five PMBC samples whose viability is <75% (CMD). PBMCs from both groups were seeded in 6-well plates in presence of BM. After 48 hours, cell death is measured by Annexin V I Hoechst staining. % Cell death is calculated as 100 minus % live cells (viable cells (negatively stained, lower left quadrants). Results represent the mean of four samples per group. Error bars represent SEM, P values are calculated using Welch’s t-test, P value < 0.05 indicates statistical significance. Figure 2C, PBMCs from both groups (CMD and CMI) were grown in CM and treated with increasing doses of venetoclax (ABT-199) (0 nM, 1 nM, 5 nM) for 48 hours. Cell viability is measured using Cell Titer-Gio (percentage relative to DMSO). Figure 2D, IC50 values of venetoclax (ABT-199) segregate based on CM dependence (CMD, n=9 and CMI, n=7). Results represent the mean per group. Error bars represent SEM. P values are calculated using Welch’s t-test. P value < 0.05 indicates statistical significance. Figure 2E, Upper, Western blot analysis of w hole cell extracts using the MCL1 antibody from both CLL groups. Actin was used as loading control. Figure 2E, Lower, Densitometry analysis was performed for images shown in upper panel. P values were calculated between the two groups using Welch’s t-test. P value = 0.0004 for the comparison of CMD vs CMI samples. Figure 2F, Upper, heatmap of BH3 profile performed on freshly isolated CLL-PBMCs, 5 patient samples derived from each group (CMD, patient # 1, 2, 3, 4 and 7 and CMI, patient # 15, 20, 21, 22 and 23 as defined in Figure 2A) using BIM and BAD peptides. Percent Cytochrome C release (indicative of MOMP) is calculated for each BH3 peptide treatment relative to maximum value of negative control (DMSO). Figure 2F, Lower, graphs showing difference in cytochrome C release in response to BIM and BAD peptides between both groups. Means are depicted as horizontal bars + / - SE error bars, P values were calculated using Welch’s t-test, P value < 0.05 indicate statistical significance. Figure 2G, Left, PBMCs are cultured in BM for 24 hours and their cell viability’ is measured using Cell Titer-Gio. Percent Cell viability is calculated relative to Day 0 (100%). IgVH status of the 23 samples is compared with their viability. Figure 2G, Right, table showing the odds ratio is ~ 17: 1 between IgHV-U given CMI status as opposed to IgHV-M with a Fisher exact test, P value = 0.01 indicates statistical significance.

[0059] Figure 3 illustrates monocytes and T-cells do not influence CLL survival. Figure 3A. CLL patients’ PBMCs n=5 from both groups are stained for either CD3+ or CD14+ and their representation compared. Figure 3B. CD 19+ cells are isolated from CLL PBMCs from both groups, n = 3. PBMCs and CD 19+ B-CLL cells were grown in RPMI for 48 hours. Cell viability was compared between PBMCs and purified CD 19+ B-CLL cells. Figure 3C. Upper, hierarchical clustering of gene expression data of CM dependent vs. CM independent CLL (n = 5 each group) using Gene Cluster 3.0. Lower, PC A analysis of different CM dependent and independent samples. Figure 3D. Heatmap showing differentially expressed genes between CM dependent vs. CM independent CLL (FDR<0.05, FC>2). Figure 3E. Signaling pathways enriched for the differentially expressed genes in (B) using Enrichr and the Molecular Signatures Database (MSigDB). Figure 3F. Relative expression of genes in RAS signaling pathway and Focal adhesion.

[0060] Figure 4 illustrates CM-independent CLL showed high FLT3 receptor expression and were preferentially sensitive to FLT3 inhibition. Figure 4A. Left, FLT3 gene expression assessed by RT-qPCR. Data represent mean + / - SEM for analysis of CLL patients’ PBMCs from both groups (n = 5). P values were calculated using Welch’s t-test, P < 0.05 indicates statistical significance. Figure 4A. Right, surface expression of FLT3 was determined by flow cytometry analysis. PBMCs from each CLL group (n = 5) is stained for FLT3. P values were calculated using Welch’s t-test, P < 0.05 indicates statistical significance. Figure 4B. Cell viability of primary CLL samples (n = 5, each group) following 72 hrs of FLT3 inhibitor treatment. CLL PBMCs are cultured in BM and treated with either gilteritinib or quizartinib for 72 hrs. Cell viability is measured using Cell Titer-Gio. Percent Cell viability is calculated relative to untreated (100%). P-value is determined by two-tailed t-test.

[0061] Figure 5A illustrates that CLL samples undergo little spontaneous cell death when cultured in BM with CM supplementation. Figure 5B is a heatmap of baseline profile performed on freshly isolated CLL-PBMCs, 4 patient samples derived from each group (CMD, patient # 1, 2, 3 and 4 and CMI, patient # 15, 20, 22 and 23 as defined in Figure 2A). Percent Cytochrome C release (indicative of MOMP) is calculated relative to maximum value of negative control (DMSO).

[0062] Figure 5C compares CLL survival when grown in CM derived from HS-5 stroma cell line with CLL cells that are cocultured directly with HS-5 cells.

[0063] Figure 5D illustrates CMI expressed higher IL8 mRNA levels compared to CMD.

[0064] Figures 5E and 5F illustrate CMI samples showed increased sensitivity to FLT3 inhibitors compared to CMD samples.

[0065] Materials and methods

[0066] CLL patient samples and cell purification:

[0067] Peripheral blood from untreated CLL patient samples with informed consent was obtained according to the guidelines and regulations of Dana-Farber Cancer Institute Review Board. Peripheral blood mononuclear cells (PBMCs) from blood samples were isolated using Ficoll-Paque density gradient centrifugation. Samples were viably frozen using fetal bovine serum (FBS, Sigma- Aldrich) with 10% dimethyl sulfoxide (DMSO, Sigma- Aldrich) and stored in liquid nitrogen until use.

[0068] Conditioned media (CM) preparation and CLL cell culture

[0069] Stromal NKTert cells (Riken cell bank) were cultured in RPMI 1640 medium with 10 % FBS. 1 % penicillin-streptomycin (Invitrogen) and 2.05 mM L-glutamine. Cells were kept in a humidified 5 % CO2 incubator at 37°C. After 48 hours, the supernatant (CM) was collected, filtered, and stored at -20°C. Primary CLL cells were cultured in CM and treated with drugs for different experiments.

[0070] Cell viability testing

[0071] Cell viability was measured using CellTiter-Glo reagent (Promega) according to manufacturer's instructions.

[0072] Cytokine array

[0073] Cytokine array was performed using Proteome Profiler Human Cytokine Array Kit (R&D) according to the manufacturer’s protocol.

[0074] IL-6. IL-8 and CCL2 ELISA

[0075] Protein levels of IL-6, IL-8 and CCL2 in CM were measured using the DuoSet ELISA kit for IL6, IL8 and Human CCL2 / MCP1 (R&D) according to the manufacturer’s protocol. BH3 profiling Viably frozen CLL cells were thawed and stained with live / dead fixable Zombie-NIR dye (1 : 100, BioLegend). BH3 profiling was performed as previously described (Ryan et al., Biol. Chem., 2016 Jul, 397(7):671-8) where CLL cells were exposed to synthetic BH3 peptides and 0.002% digitonin in MEB2 buffer (150 mM mannitol, 10 mM HEPES-KOH pH 7.5, 150 mM KC1, 1 mM EGTA, 1 mM EDTA, 0. 1 % BSA and 5mM Succinate) for 1 hour at 25°C. After 1 hour incubation, cells were fixed with 8 % formaldehyde for 15 minutes followed by neutralization with N2 buffer (1.7 M Tris, 1.25 M Glycine pH 9.1) for 5 mins. After neutralization, cells were stained with anti-cytochrome c Al exafluor-488 antibody (Biolegend) in staining buffer (10 % BSA, 2 % Tween 20, PBS) to measure the sensitivity to BH3 peptides via flow cytometry.

[0076] Dynamic BH3 profiling

[0077] Viably frozen CLL cells were thawed and cultured in CM ex-vivo with or without drugs. After 20 hours, cells were collected and BH3 profile performed using different synthetic BH3 peptides. FACS data were analyzed and delta priming was calculated (delta priming = Cytochrome C releasedrug- Cytochrome C releasedmso).

[0078] Western blot analysis

[0079] Whole-cell extracts were prepared by lysing CLL PBMC samples using RIPA lysis buffer (Sigma Aldrich) with protease inhibitor (Millipore) and phosphatase inhibitor (Sigma Aldrich). Protein concentration was quantified using Coomassie Protein Assay Reagent (Pierce). Protein ly sates were separated through SDS-polyacrylamide gels (4 - 12 %) and transferred to PVDF membrane (Millipore). The membranes were blocked with 5 % milk powder in 0.1 % Tween20 in 1 x PBS (PBS-T) for 1 hour followed by primary antibody incubation. Images were obtained by exposing membrane to ImmunoCruz Western Blotting Luminol Reagent (Santa Cruz Biotechnology7, Inc.).

[0080] Antibodies

[0081] The following antibodies were used in this study: PARP (9542S, cell signaling). Caspase 3 (9665S, cell signaling). Actin (58169S, cell signaling). Neutralizing antibodies used in this study: Human IL-8 MAB (CL 6217) from R&D system, Human IL-6 MAB (CL 6708) from R&D system, Human CCL2 / MCP-1 MAB (CL 23007) from R&D system. RT-PCR

[0082] The pellets from CLL PBMC samples were resuspended in Trizol. The RNA from these samples were harvested using the Zymo Direct-zol RNA Microprep kit. cDNA was prepared by Bio-rad clear gDNA iScript™ gDNA Clear cDNA Synthesis Kit, as per the manufacturer’s protocol. Quantitative RT-PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a QuantStudioTM Flex 6 System platform. Gene of interest amplification was normalized using the b-actin or Gapdh expression and alterations among sample groups were calculated using the 2-AA cycle threshold method. IL8 primers (forward, 5 ’ -CGGAAGGAACC ATCTC ACTG-3 ’ ; reverse, 5 ’ -CC AGTTTTCCTTGGGGTCC A-3 ’ ). FLT3 primers (forward, 5 -CTGCCGCTGCTCGTTGTT-3’; reverse, 5’- TGATGATGACTTCCCC ACTGA-3 ’) RNA-seq

[0083] Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200 ng of purified total RNA according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to manufacturer's protocols. Uniquely dual indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NovaSeq 6000 with paired-end 50bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Sequenced reads were aligned to the UCSC hg!9 reference genome assembly and gene counts were quantified using STAR (v2.7.3a) (Dobin et al., Bioinformatics, 2013;29(l): 15-21). Differential gene expression testing was performed by DESeq2 (v 1.22.1) (Love et al., Genome Biol., 2014; 15(12):550). RNAseq analysis was performed using the VIPER snakemake pipeline (Cornwell et al., BMC Bioinformatics , 2018; 19(1): 135).

[0084] Data analysis and statistics

[0085] Statistical analyses were performed using GraphPad Prism 6, unless otherwise specified in Methods.

[0086] Example 1 - Conditioned media derived from NKTert stroma cells supports CLL survival

[0087] While CLL cells undergo spontaneous cell death in ex vivo culture, co-culture with bone marrow derived NKTert stromal cells is known to inhibit this death. However, it was not known whether cell-cell contact is necessary' for survival enhancement, or whether humoral elements in the co-culture supernatant suffice. An experiment was conducted to see if CM from the supernatant of NKTert cells could substitute for NKTert co-culture to maintain CLL survival. Survival of CLL cells maintained in CM (Figure 1A) was compared with that of CLL cells grown in co-culture with NKTert cells for four days. No significant difference in cell viability between them was seen until day 3 (Figure IB). Similarly, CLL survival when grown in CM derived from HS-5 stroma cell line was compared with CLL cells that are cocultured directly with HS-5 cells. Again, no significant difference in cell viability was seen (Figure 5C). CLL cells grown in CM supplement showed less cell death compared to those grown in RPMI complete media alone (Basal Media, BM) (Figure 1C).

[0088] Example 2 - A subgroup of apoptosis resistant CLL cells have a reduced requirement for CM support

[0089] Surprisingly, the inventors found that -39 % (9 / 23 cases) CLL samples undergo little spontaneous cell death even when cultured in BM without CM supplementation (Figures 2A and 2B and Figure 5A). To define if there were any functional differences between those primary cells that survived without CM (CMI) vs the primary samples that depend on CM for ex vivo survival (CMD), it was checked if there is a difference in venetoclax sensitivities between these two groups, since venetoclax-based therapy has improved outcomes even in high risk CLL patient populations. CMI samples (n = 7) were less sensitive to venetoclax treatment than the CMD group (n = 9) (P value = 0.0002, Figures 2C and 2D) even when grown in identical CM.

[0090] Since CMI cells were less sensitive to venetoclax, BH3 profiling was performed in a subset of 5 patient samples from each group to test if differences in mitochondrial apoptotic priming correlated with differences in venetoclax sensitivity. BH3 profiling measures cytochrome c release from the mitochondria as a result of mitochondrial outer membrane permeabilization (MOMP) in response to synthetic BH3 peptides (Certo et al., Cancer Cell, 2006;9(5):351-365). CMI samples showed decreased MOMP in response to BIM (p=0.014), PUMA (p= 0.029) and BAD (p=0.0036) peptides compared to CMD specimens, consistent with decreased mitochondrial apoptotic priming (Figure 2F and Figure 5B).

[0091] Given the influence of IGHV mutational status on BCR signaling, it w as also checked whether one could categorize CMI CLL cells vs CMD CLL patient samples based on IGHV. We found that 7 / 9 (-78%) CMI CLL had unmutated-IGHV and 12 / 14 (86%) CMD CLL patient harbor-mutated IGVH (Figure 2H).

[0092] Example 3 - CMI CLL showed high FLT3 receptor expression and were preferentially sensitive to FLT3 inhibition.

[0093] A further experiment investigated whether CD 14+ cells in primary CLL-PBMC samples are increased in number and required for survival of the CMI samples. To the contrary, there were slightly fewer CD14+ monocytes and CD3+ T-cells in CMI CLL cells compared to CMD CLL cells (Figure 3A). To evaluate if the presence of non-CLL accessory cells in the PBMC samples supported survival in BM, CD19+ CLL cells from both groups’ PBMCs were isolated and the viability' of the leukemia cells to that of total CLL -PBMCs when cultured in BM for 2 days was compared. The viability of CLL cells grown in presence of accessory cells (including CD14+ and CD3+ cells) was similar to that of the purified CD19+ CLL cells alone (Figure 3B) suggesting that CM-independence is a feature intrinsic to CLL cells.

[0094] In a further experiment, gene expression between CMD and CMI CLLs was compared by RNAseq analysis (n = 5 for each group). As shown in Figure 3C, unsupervised hierarchical clustering revealed clear partition of the two groups into different clusters, and CMI CLLs are more likely to be IGHV unmutated (4 out of 5). Principal Component Analysis (PCA) further revealed a tight clustering of CMD CLLs, while CMI CLLs w ere more dispersed, indicating higher levels of heterogeneity7amongst those samples. As shown in Figure 3D, altogether 97 upregulated and 55 downregulated genes were identified in CMI vs. CMD samples (FDR<0.05, FC>2). Differentially expressed genes were highly enriched for Focal adhesion and Ras signaling pathways in CMI samples compared to CMD samples. (Figure 3E). 5 out of the 6 significantly altered genes (KSR2, PDGFA, RASAL2, FLT3, INSR) involved in Ras signaling were upregulated (Figure 3F). Upregulation of FLT3 receptor expression in CMI samples compared to CMD group was also observed, indicating activation of the Ras signaling pathway (Figure 5E).

[0095] Since RNA-seq analysis revealed upregulation of FLT3 in the CMI group, an experiment was conducted to further validate if FLT3 is differentially expressed between these groups. CMI samples (n = 5) demonstrated very7high levels of FLT3 mRNA and surface receptor tyrosine kinase protein compared to CMD samples (n = 5) (Figure 4A).

[0096] A further experiment tested if it would be possible to target the CMI group with different selective FLT3 inhibitors (quizartinib and gilteritinib). Compared to CMD samples, CMI samples showed increased sensitivity to both FLT3 inhibitors (Figure 4B and Figure 5E). This demonstrates that FLT3 inhibitors may be effective in the treatment of the CMI subset of CLL.

[0097] The foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0098] The patent and scientific literature referred to herein establishes the knowledge that is available to those with ordinary skill in the art. All documents cited herein are hereby incorporated by reference.

Claims

CLAIMS:

1. A method of treating CLL comprising the administration of a therapeutically effective amount of a FLT3 inhibitor and, optionally, one or more additional agents which are suitable for the treatment of CLL to a patient in need thereof.

2. A FLT3 inhibitor and, optionally, one or more additional agents which are suitable for the treatment of CLL for use in a method of treating CLL comprising the administration of said FLT3 inhibitor and, optionally, said one or more additional agents which are suitable for the treatment of CLL to a patient in need thereof.

3. Use of a FLT3 inhibitor and, optionally, one or more additional agents which are suitable for the treatment of CLL in the manufacture of a medicament for the treatment of CLL in a patient in need thereof.

4. The method of claim 1, compound for use of claim 2 or use of claim 3, wherein the patient has been previously identified as suffering from CLL which is CM-independent.

5. The method of claim 1, compound for use of claim 2 or use of claim 3, wherein the patient has been previously identified as suffering from CLL which is CCL2 independent.

6. The method of claim 1, compound for use of claim 2 or use of claim 3, wherein the patient has been previously identified as suffering from CLL which is resistant to treatment with a BCL2 antagonist.

7. The method, compound for use, or use of any preceding claim, wherein the FLT3 inhibitor is the sole active ingredient used to treat CLL.

8. The method, compound for use, or use of any preceding claim, wherein the FLT3 inhibitor is selected from the group consisting of gilteritinib, quizartinib, midostaurin, luxeptinib, sorafenib, tandutinib, lestaurtinib, imidacrine and crenolanib.

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