ALK inhibitors for the treatment of ALK-negative cancers and plasma cell-mediated diseases

Abstract

The present invention provides a method of treating ALK-negative / LTK-positive cancer in a subject, the method comprising administering to the subject a pharmaceutically effective dose of a linear inhibitor of ALK. The present invention is particularly useful for treating multiple myeloma, including proteasome inhibitor-resistant multiple myeloma.

Description

[Technical field]

[0001] The present invention relates to the treatment of anaplastic lymphoma kinase (ALK)-negative and leukocyte tyrosine kinase (LTK)-positive cancers in humans. Neg LTK Pos The present invention provides linear ALK inhibitors for use in treating ALK-negative, LTK-positive plasma cells (target cells). The present invention also provides linear ALK inhibitors for use in treating plasma cell mediated diseases in humans characterized by ALK-negative, LTK-positive plasma cells. The plasma cell mediated diseases treated according to the present invention may in particular be autoimmune diseases characterized by the production of autoantibodies.

[0002] Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that belongs to the insulin receptor superfamily. In healthy adults, ALK protein is expressed at low levels only in the central nervous system (Pulford et al. Blood 89:1394-1404,1997). However, as reviewed by Hallberg & Palmer, 2016 (Annals of Oncology 27 (Supplement 3): iii4-iii15), aberrant ALK expression has been reported in many cancers. ALK fusion proteins have been reported to be expressed in several cancers. ALK has been found to fuse with various proteins, notably NPM (e.g., in anaplastic large cell lymphoma) and EML4 (e.g., in non-small cell lung cancer (NSCLC)). Oncogenic ALK fusions involve the kinase domain of the ALK protein and are ligand-independent (i.e., the ALK domain is constitutively active). Other mechanisms of ALK-mediated oncogenesis have also been identified: point mutations in the full-length ALK gene have been reported, particularly in neuroblastoma; and overexpression of full-length ALK has been reported in several tumor types.

[0003] ALK Pos The cancer is an ALK-expressing cancer, but not one that has any form of ALK. PosALK inhibitors are widely known for the treatment of cancer. For example, WO 2009 / 126154 and WO 2010 / 002655 disclose triazine and pyrimidine derivatives for use in the treatment of ALK-mediated conditions, particularly ALK-mediated cancers. The first ALK inhibitor approved for cancer treatment was crizotinib, which inhibits ALK. Pos It was approved for the treatment of NSCLC in 2011. A number of other ALK inhibitors have been approved for this treatment, including ceritinib, alectinib, and brigatinib. Ceritinib was first disclosed in WO 2008 / 073687.

[0004] The present invention provides a new use of known ALK inhibitors. Neg LTK Pos The invention also provides an ALK inhibitor for the treatment of cancer. Neg LTK Pos The present invention provides an ALK inhibitor for the treatment of plasma cell-mediated diseases characterized by plasma cells that are ALK-positive. Leukocyte tyrosine kinase (LTK) is an endoplasmic reticulum (ER)-resident receptor tyrosine kinase that regulates trafficking out of the ER (Centonze et al., Journal of Cell Biology 218(8): 2470-2480, 2019). The present inventors have demonstrated that LTK plays an important role in protein homeostasis.

[0005] Proteostasis involves the synthesis and transport of nascent proteins on the one hand, and the degradation of misfolded proteins on the other hand. The biosynthetic and degradative parts of proteostasis are highly interconnected, forming the so-called proteostasis network. The endoplasmic reticulum is the main site of proteostasis and is equipped with various control systems that sense and respond to proteome imbalances. The accumulation of misfolded proteins activates three endoplasmic reticulum-resident transmembrane proteins (IRE1, ATF6, and PERK), resulting in the induction of the unfolded protein response (UPR). The UPR not only increases the ability of the endoplasmic reticulum to fold proteins, but also stimulates the proteasomal degradation of terminally misfolded proteins. The UPR can therefore be referred to as an autoregulatory process of the proteostasis network. While misfolded proteins leave the ER bound for degradation compartments, correctly folded secretory proteins leave the ER in COPII carriers that form at specialized regions called endoplasmic reticulum exit sites (ERES).While the adaptive response to misfolded proteins is fairly well understood, much less is known about the equivalent mechanisms that sense the folded protein load and respond by modulating ERES.

[0006] It is shown herein that LTK responds to secretory load and regulates the secretory capacity of endoplasmic reticulum, making endoplasmic reticulum a regulatory point in the protein homeostasis network.Thus, LTK represents a target in the biosynthetic part of the protein homeostasis network that can be targeted for the treatment of diseases characterized by excessive protein secretion.Many cancers are characterized by high levels of protein secretion (hypersecretion) and show high dependency on protein homeostasis mechanisms.Certain plasma cell-mediated diseases, such as autoimmune disorders, are also characterized by protein (antibody) hypersecretion by plasma cells, plasmablasts, and their precursors.

[0007] A specific example of a disease state characterized by protein hypersecretion is multiple myeloma. Multiple myeloma is a cancer of clonal plasma cells. The cancerous cells are antibody-producing cells, and multiple myeloma is characterized by antibody hypersecretion. The importance of protein homeostasis in multiple myeloma is evident from the clinical success of proteasome inhibitors that block the degradation part of the protein homeostasis network (Moreau et al., Blood 120(5): 947-959, 2012). Recent advances in the treatment of multiple myeloma have led to improved survival, but as of 2011, the overall 5-year survival rate for multiple myeloma was only 49% (Kazandjian, Seminars in Oncology 43(6): 676-681, 2016), and although therapies may result in disease remission, they are not considered to be curative. Thus, new therapies are needed.

[0008] Another example of a pathology characterized by cells exhibiting protein hypersecretion is chronic lymphocytic leukemia (CLL), a cancer characterized by the clonal proliferation of B lymphocytes in lymph nodes, spleen, or bone marrow. CLL blast cells (blast cells) that proliferate in the cancer tissue (so-called pseudofollicles) secrete monoclonal antibodies before undergoing cell division (Darwiche et al., Frontiers in Immunology 9:683, 2018).

[0009] Other examples of conditions characterized by protein hypersecretion are autoimmune diseases associated with the production of autoantibodies by dysregulated plasma cells. One such condition is lupus (also known as systemic lupus erythematosus (SLE)). In lupus, dysregulated B-cell differentiation leads to the development of plasma cells that produce autoantibodies against cell nuclear components, such as antinuclear antibodies (ANA). Particularly common targets of autoantibodies in lupus include double-stranded DNA and ribonucleoproteins. Another example of such an autoimmune disease is immune thrombocytopenia (ITP), in which dysregulated plasma cells produce antibodies against platelet surface structures, causing platelet deficiency and, as a result, blood clotting defects. Effective treatments for such autoimmune diseases are needed.

[0010] The inventors have previously shown that certain known ALK inhibitors are effective in inhibiting LTK (Centonze et al., supra). The inventors now demonstrate that targeting LTK with a clinically approved ALK inhibitor sensitizes hypersecretory cells, such as multiple myeloma cells and proliferating CLL blasts, to endoplasmic reticulum stress and induces apoptosis. Similarly, proapoptotic effects occur in dysregulated plasma cells that secrete high levels of autoantibodies in various autoimmune diseases. Accordingly, ALK inhibitors inhibit ALK Pos Previously thought to be of fairly limited utility in treating cancer, the present invention provides a novel method for the treatment of ALK Neg LTK Pos Cancer (especially ALK Neg LTK Pos Multiple myeloma and ALK Neg LTK Pos This invention provides novel uses for some such agents in the treatment of chronic lymphocytic leukemia (CLL), and plasma cell-mediated diseases characterized by cells that are ALK- / LTK+. The inventors show that the effect of ALK inhibitors is independent of ALK expression, since LTK+ALK- knockdown cell lines or ALK- knockdown cell lines remain sensitive to ALK inhibition.

[0011] Thus, in a first aspect, the present invention provides a linear inhibitor of ALK for use in treating cancer or a plasma cell mediated disease in a subject, wherein the cancer or plasma cell mediated disease is characterised by cells that are ALK-negative and LTK-positive.

[0012] In a similar aspect, the present invention provides a method of treating cancer or a plasma cell mediated disease in a subject, wherein the cancer or plasma cell mediated disease is characterized by cells that are ALK-negative and LTK-positive, the method comprising administering to the subject a linear inhibitor of ALK in an amount effective to treat the cancer.

[0013] Also provided is a method of diagnosing and treating ALK-negative cancer in a subject, the method comprising: (i) diagnosing cancer in said subject; (ii) examining the cancer for expression of ALK and LTK; and (iii) if the cancer is found to be ALK negative and LTK positive, administering to the subject a linear inhibitor of ALK in an amount effective to treat the cancer.

[0014] Also provided herein are methods of diagnosing and treating ALK-negative cancer in a subject, the methods comprising: (i) diagnosing cancer in said subject; (ii) determining that the cancer is ALK negative and LTK positive; and administering to the subject a linear inhibitor of ALK in an amount effective to treat the cancer.

[0015] In another aspect, the invention provides the use of a linear inhibitor of ALK in the manufacture of a medicament for treating cancer or a plasma cell mediated disease in a subject, wherein the cancer or plasma cell mediated disease is characterized by cells that are ALK-negative and LTK-positive.

[0016] In another aspect, the present invention provides a linear inhibitor of ALK for use in treating proteasome inhibitor-resistant cancer.The proteasome inhibitor-resistant cancer is preferably proteasome inhibitor-resistant multiple myeloma.The proteasome inhibitor-resistant cancer may be LTK-positive cancer, particularly LTK-positive multiple myeloma.

[0017] As described in more detail below, the subject may be any mammalian subject, however, in certain preferred embodiments of this aspect, the subject is a human subject.

[0018] As mentioned above, the present inventors have discovered that some known ALK inhibitors (also referred to herein as inhibitors of ALK) are also effective in inhibiting LTK. In particular, as shown in the Examples, they have found that linear ALK inhibitors are effective in inhibiting LTK. However, they have found that cyclic ALK inhibitors are ineffective. Although ALK and LTK are related proteins (their cytoplasmic kinase domains are 79% identical in humans, Centonze et al., supra), it could not have been expected that some ALK inhibitors would show cross-specificity for LTK, while others would not.

[0019] As described herein, an ALK inhibitor is a compound that can interact with ALK (especially human ALK) and reduce or neutralize its activity. The UniProt accession number of human ALK is Q9UM73. An ALK inhibitor can inhibit the activity of both full-length, wild-type ALK and ALK fusion proteins. As described in Chand et al. (Disease Models & Mechanisms 6: 373-382, 2013, which is incorporated herein by reference), numerous methods are known for testing compounds for ALK inhibitory activity. For example, ALK activation requires autophosphorylation of Tyr1278. Inhibition of ALK activity thus inhibits Tyr1278 autophosphorylation, which can be detected by immunoblot using anti-phosphorylated ALK (Y1278) antibody. Such antibodies are commercially available (e.g., antibody #3710, Cell Signaling Technology, USA). Thus, to determine whether a compound has activity in inhibiting ALK (i.e., is an ALK inhibitor), the compound of interest can be applied to cells expressing human ALK, the cells lysed, and ALK autophosphorylation at Tyr1278 analyzed.

[0020] Other methods for testing ALK inhibition are known, for example assaying ALK autophosphorylation at Tyr1604 and / or phosphorylation of ALK substrates such as ERK and STAT3, as described in Chand et al. (see above).

[0021] Several ALK inhibitors are known in the art, and to date, six have been approved for medical use: crizotinib, ceritinib, alectinib, brigatinib, entrectinib, and lorlatinib. Another known ALK inhibitor is berizatinib.

[0022] Said ALK inhibitor for use as described herein is also effective for inhibiting LTK, particularly human LTK. The UniProt accession number of human LTK is P29376. "Effective for inhibiting LTK" means that the inhibitor can interact with LTK, particularly human LTK, and reduce or abolish the activity of LTK. Methods for testing compounds for LTK inhibitory activity are known and are described in the examples below. In particular, LTK activation requires autophosphorylation of Tyr672, which is equivalent to the autophosphorylation of ALK at Tyr1278. Many antibodies that recognize phosphorylated ALK (Y1278) also recognize phosphorylated LTK (Y672). Suitable antibodies for detecting phosphorylated LTK (Y672) are commercially available (e.g., antibody D59G10, Cell Signaling Technology). Thus, to determine whether a compound has LTK inhibitory activity (i.e., is an inhibitor of LTK), the compound may be applied to cells expressing human LTK, the cells lysed and LTK autophosphorylation at Tyr672 analyzed.

[0023] As mentioned above, the inventors have discovered that linear ALK inhibitors also have activity in inhibiting LTK, while cyclic ALK inhibitors have no effect on LTK. By "linear" ALK inhibitors, we mean ALK inhibitors that do not have a central ring (i.e., a central ring from which all functional groups emanate) that serves as a molecular core. Such rings are referred to herein as cyclic molecular cores. Linear ALK inhibitors may nevertheless contain cyclic functional groups, such as phenyl groups. However, in this example, not all functional groups and side chains extend from the same cyclic structure (or part thereof). In other words, the term "linear ALK inhibitors" does not include molecules that are cyclized in the sense of having a "circular" structure (i.e., a circular backbone) that does not have distinct defined ends. ALK inhibitors that contain a cyclic molecular core are cyclic ALK inhibitors, or, in other words, cyclized or circular ALK inhibitors.

[0024] An example of a cyclic ALK inhibitor is lorlatinib, which has the structure shown in formula I. As shown, lorlatinib contains a 12-membered ring, which forms the core of the molecule. With this core ring, lorlatinib is a cyclic ALK inhibitor. Lorlatinib has been found to have no LTK inhibitory activity. [ka]

[0025] Linear ALK inhibitors have a structure that lacks a cyclic molecular core. Rather, they contain many functional groups linked together in series. Examples of linear ALK inhibitors include crizotinib, ceritinib, brigatinib, ensartinib, alectinib, and entrectinib. The structure of crizotinib (3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine) is shown in formula II; the structure of ceritinib (5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine) is shown in formula III; the structure of brigatinib (5-chloro-4-N-(2-dimethylphosphorylphenyl)-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]pyrimidine-2,4-diamine) is shown in formula IV; The structure of ensartinib (6-amino-5-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-N-[4-[(3R,5S)-3,5-dimethylpiperazine-1-carbonyl]phenyl]pyridazine-3-carboxamide) is shown in formula V; the structure of alectinib (9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile) is shown in formula V; the structure of entrectinib (N-[5-[(3,5-difluorophenyl)methyl]-1H-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide) is shown in formula IV. [ka] [ka] [ka] [ka] [ka] [ka]

[0026] As can be seen in Formulas II-VII, the linear ALK inhibitors lack a cyclic molecular core: While all of the exemplified linear ALK inhibitors contain a cyclic group (e.g., a phenyl group), none of them form a central ring from which all functional groups emanate.

[0027] In non-linear, "cyclic" ALK inhibitors, the cyclic molecular core may be the largest cyclic group in the ALK inhibitor. In particular, the number of members of the cyclic molecular core may be at least 2 greater than the number of members of other cyclic groups in the molecule. In other words, the cyclic molecular core may be at least 8 membered rings, such as 9 membered rings, 10 membered rings, 11 membered rings, or 12 membered rings. The cyclic molecular core of lorlatinib is a 12 membered ring. As mentioned above, a linear ALK inhibitor is an ALK inhibitor that does not have a cyclic molecular core.

[0028] The ALK inhibitor according to the present invention is Neg And LTK Pos The present invention is for use in the treatment of cancer or plasma cell-mediated diseases characterized by cells that are ALK. Neg And LTK Pos Cancers characterized by cells that are ALK-positive are cancers in which the malignant cells that make up the cancer express LTK but do not express ALK. Neg (ALK negative and LTK) Pos (LTK positive).

[0029] As used herein, ALK PosBy (ALK positive) cancer is meant a cancer that expresses any form of ALK protein, including as a wild-type protein, an ALK fusion protein, or a mutated full-length protein (including, for example, a gain-of-function mutation). Methods for determining whether a cancer expresses ALK are standard in the art and are currently routinely used to determine the status of ALK expression for cancers for which ALK inhibitor therapy has been approved, particularly NSCLC.

[0030] Such methods are typically performed on cancer biopsy samples. An example of a suitable method for detecting ALK expression is immunohistochemistry (IHC). IHC kits for detecting ALK expression have received regulatory approval and are commercially available. An example of such a kit is the Ventana ALK (D5F3) CDx Assay (Roche, Switzerland), which can be used to detect ALK expression. Other such kits may also be used.

[0031] In certain embodiments, a cancer that tests positive in a regulatory approved ALK expression test is considered ALK positive, and a cancer that tests negative in such an ALK expression test is considered ALK negative. Pos ALK Neg It can be judged normally.

[0032] Thus, ALK Neg The cancer may be a cancer that is determined to be ALK negative when cancer tissue is analyzed for expression of ALK. Neg The cancer may be one in which ALK expression is not detected by IHC. For example, an ALK-negative cancer may be one in which a regulatory approved assay for detecting ALK expression gives a negative result. In certain embodiments, ALK Neg The cancer is one that tests negative by the Ventana ALK (D5F3) CDx Assay.

[0033] ALK gene rearrangement may be detected directly at the genomic level, for example, using fluorescent in situ hybridization (FISH). In particular, break-apart FISH may be used to detect ALK gene rearrangement, and suitable break-apart FISH kits are commercially available (e.g., Vysis ALK Break Apart FISH Probe Kit, Abbott Molecular, USA, product number 06N38-023) or can be designed by a skilled technician. Cancers exhibiting ALK gene rearrangement at the DNA level are herein referred to as ALK. Pos As mentioned above, the present invention relates to the treatment of ALK cancer.

[0034] Similarly, L.T.K. Pos The cancer is a cancer with detectable levels of LTK expression. Similar to the detection of ALK, the expression of LTK can be detected using methods standard in the art. Such methods may typically be performed on biopsy samples. For example, immunohistochemistry (e.g., immunofluorescence staining) may be used to directly detect the expression of LTK protein. Reverse transcriptase PCR (RT-PCR) may be used reliably to detect the expression of LTK at the mRNA level. Primers suitable for use in LTK PT-PCR can be designed by a skilled technician. Preferably, qPCR is used to detect the expression of LTK. Primers suitable for use in LTK qPCR can be designed by a skilled technician or are commercially available (e.g., LTK QuantiTect primer assay, Qiagen, catalog number QT00219877).

[0035] Thus, according to the present invention, cancer may be diagnosed in a subject using standard methods in the art. The cancer may then be examined for expression of ALK and LTK. A biopsy sample may be obtained for ALK and LTK expression testing. When a biopsy sample is used to diagnose cancer, the same biopsy sample may be used for ALK and LTK expression testing, or a different biopsy sample may be used. The biopsy sample may be obtained by any means appropriate for the type of cancer. For example, the biopsy sample may be obtained by open biopsy, e.g., excision biopsy sample. Alternatively, the sample may be obtained by needle biopsy. In the case of blood cancers, such as multiple myeloma or CLL, the biopsy sample may be obtained by bone marrow biopsy (myeloma or CLL) or may be a blood sample (CLL). A skilled physician can obtain an appropriate biopsy sample.

[0036] The biopsy sample may be a liquid biopsy sample, e.g. obtained from blood. In particular, circulating tumor cells may be isolated and analyzed for ALK and LTK expression, as described above. A liquid biopsy method for the diagnosis and characterization of multiple myeloma is disclosed in Kamande et al. (Integrative Biology 10(2): 82-91, 2018), which may be used to examine for ALK and LTK expression, as described above. If the cancer being examined is ALK Neg LTK Pos If found to be the case, they may be treated with an ALK inhibitor according to the present invention.

[0037] Thus, using the above techniques, cancers that are ALK Neg And LTK Pos Alternatively, in some instances, a cancer may be determined to be ALK positive based on known characteristics of the cancer. Neg And LTK Pos In this way, if circumstances permit, it may be determined without experimental confirmation.

[0038] As used herein, the term "cancer" is used in its usual sense in the art, i.e., a malignant neoplastic condition. The cancers treated according to the present invention may be any type of cancer, including both solid and hematological cancers. ALK Neg And LTK Pos Any cancer that is a cancer may be treated according to the present invention.

[0039] In one embodiment, the cancer to be treated according to the present invention is ROS1 negative (ROS1 Neg ROS1 is a tyrosine kinase related to ALK. Its role in normal development is unclear, but it is known to be aberrantly expressed in some cancers. Many ALK inhibitors have demonstrated activity in inhibiting ROS1, and such ALK inhibitors (e.g., crizotinib) inhibit ROS1. Pos It has been found to be useful in the treatment of cancer. The UniProt accession number for human ROS1 is P08922. In the context of cancer, ROS1 has been found to arise as a result of gene rearrangements and to be expressed in fusion proteins. Several oncogenic ROS1 fusion proteins are known in which ROS1 is constitutively active, including fusions with FIG, CD74, TPM3, SDC4, and SLC34A2, among others.

[0040] Some ROS1-negative cancers do not exhibit detectable expression of ROS1. However, ROS1 expression is found in healthy adult human tissues. Thus, ROS1 Neg Cancer may more preferably be defined as cancer that is not due to expression of ROS1. Neg A cancer may be defined as a cancer that does not express mutant ROS1 resulting from a gene rearrangement. Neg Cancers do not express the ROS1 fusion protein. This is called "ROS1 positive" (ROS1 Pos ) is commonly used to refer to cancers that express ROS1 fusion proteins.

[0041] The involvement of ROS1 in cancer, particularly the role of ROS1 fusion proteins and how such proteins can be detected, is described in Davies & Doebele, Clinical Cancer Research 19(15): 4040-4045, 2013. Expression of ROS1 fusion proteins may be detected using any method known in the art, such as qPCR or immunohistochemistry. Alternatively, ROS1 gene rearrangements may be detected directly at the DNA level, for example by FISH (a "break-apart" FISH assay for the detection of ROS1 gene rearrangements is described in Davies et al., Clinical Cancer Research 18(17): 4570-4579, 2012, which is incorporated herein by reference), or by next-generation genomic sequencing. Thus, the cancers treated according to the present invention include those characterized by ALK, ALK-, or ALK-like fusion proteins. Neg And ROS1 Neg And LTK Pos may be also possible.

[0042] In another embodiment, the cancer to be treated according to the present invention is NTRK fusion negative (referred to herein as NTRK-f negative, NTRK-f Neg NTRK1, NTRK2, and NTRK3 are the three NTRK genes in humans, which encode TrkA, TrkB, and TrkC, respectively. The UniProt accession number for TrkA is P04629; the UniProt accession number for TrkB is Q16620; and the UniProt accession number for TrkC is Q16288. The Trk proteins are receptor tyrosine kinases related to ALK. Similar to ALK and ROS1, Trk gene rearrangements have been found to be associated with cancer, particularly rearrangements that result in constitutively active Trk fusion proteins. Several such oncogenic Trk fusion proteins are known, including fusions of Trk proteins with TPM3, CD74, TRIM24, and ETV6.

[0043] NTRK-f NegThe cancer is one that does not express mutant Trk proteins, particularly Trk fusion proteins, resulting from gene rearrangements. Methods for detecting NTRK genes are known in the art and are discussed in Hsiao et al., Journal of Molecular Diagnostics 21(4): 553-571. Suitable methods include immunohistochemistry (utilizing pan-Trk antibodies capable of binding to TrkA, TrkB, and TrkC fusion proteins), FISH (particularly break-apart FISH), qPCR, and next-generation DNA sequencing. Thus, the cancer treated according to the present invention is one that does not express mutant Trk proteins, particularly Trk fusion proteins, resulting from gene rearrangements. Neg And NTRK-f Neg And LTK Pos In certain embodiments, the cancer to be treated according to the present invention may be ALK Neg And Ros1 Neg And NTRK-f Neg And LTK Pos (ALK Neg Ros1 Neg NTRK-f Neg LTK Pos ).

[0044] The cancer treated according to the present invention may be a cancer associated with protein hypersecretion. A cancer associated with protein hypersecretion is a cancer that has high metabolic activity and secretes significantly more protein than other cells. An example of a cancer associated with protein hypersecretion is multiple myeloma, which is associated with hypersecretion of a monoclonal immunoglobulin known as M protein. Multiple myeloma is a cancerous plasma cell that exhibits protein hypersecretion. As shown in more detail below, CLL cells also express LTK, are ALK negative, and are targeted by ALK inhibitors in the proliferative state. In the present specification, CLL cells are cancerous blast cells that exhibit protein hypersecretion. As shown in more detail below, non-cancerous plasma cells also express LTK, are ALK negative, and are targeted by ALK inhibitors.

[0045] In a particular embodiment, the cancer treated according to the present invention is multiple myeloma. Multiple myeloma (also known simply as "myeloma") is a cancer of clonal plasma cells, as described above. Multiple myeloma may be diagnosed based on the presence of M protein in the blood and / or urine. More commonly, multiple myeloma is diagnosed by performing a bone marrow biopsy to determine the percentage of plasma cells in the bone marrow. Medical imaging (e.g., x-rays, MRI scans, and PET / CT scans) may be performed to identify lytic lesions in the skeleton characteristic of multiple myeloma. Multiple myeloma may thus be identified using methods standard in the art. ALK Neg LTK Pos Multiple myeloma may be treated with an ALK inhibitor according to the present invention.

[0046] In the following examples, the ALK inhibitor Neg LTK Pos As shown in the Examples below, a significant proportion of multiple myeloma cases are associated with ALK Neg (>99.8% of multiple myeloma from 767 patients). Thus, the present invention has the advantage of being able to significantly improve LTK in multiple myeloma patients. Pos It offers a new treatment option for a subset (estimated to be more than 80% of multiple myeloma patients based on the analysis of 767 patients described below).

[0047] In another embodiment, the cancer treated according to the present invention is B-cell malignant chronic lymphocytic leukemia (CLL). Of the 107 CLL patients analyzed by the present inventors, 94 (94 / 107 (88%)) had CLL cells expressing LTK, and none had CLL cells expressing ALK. Thus, the majority of CLL patients examined expressed ALK. Neg LTK PosThe patient had cancer. CLL cells divide in lymphoid tissue or bone marrow in pseudofollicles where cancer-supporting cells activate CLL cell division. Malignant CLL cells become activated large cancer cells called proliferative blasts. In this state, CLL cells also become hypersecretory and secrete monoclonal Ig (M protein). CLL patients may have one of two forms of cancer: IgVH-mutated CLL and IgVH-unmutated CLL. Both IgVH-mutated and IgVH-unmutated forms of CLL are susceptible to killing by ALK inhibitors, and therefore both IgVH-mutated and IgVH-unmutated CLL may be treated according to the present invention. CLL cancer cells from previously treated and untreated patients are susceptible to killing by ALK inhibitors. Thus, ALK inhibitors may be used according to the present invention as a first-line treatment for CLL (e.g., newly diagnosed or newly symptomatic CLL) or for the treatment of relapsed CLL.

[0048] In another embodiment, the cancer treated according to the present invention is ALK Neg LTK Pos Hepatic cancer. Hepatic cancer refers to primary hepatic cancer, not secondary hepatic cancer that has metastasized to the liver from other parts of the body. All types of hepatic cancer, including hepatocellular carcinoma, cholangiocarcinoma, and other rare types of hepatic cancer, may be treated according to the present invention.

[0049] LTK and ALK are expressed in all mammals. Accordingly, the present invention extends to the treatment of any mammalian subject. Thus, the subject may be a livestock animal, a farm animal, or a sport animal, in addition to a human subject. The present invention relates to the treatment of ALK in humans or non-human mammals. Neg LTK Pos Examples of non-human mammals which may be treated in accordance with the present invention include dogs (canines), cats (felines), and horses (equines).

[0050] The cancer treated according to the present invention may be at any stage, such as stage I, stage II, stage III, or stage IV cancer. Thus, the cancer treated according to the present invention may be localized or may have spread throughout the body. With respect to multiple myeloma, the myeloma may be at stage 1, stage 2, or stage 3, as classified by the International Staging System. The cancer treated according to the present invention may be a recurrence of cancer, for example, the present invention may be used to treat relapsed or recurrent multiple myeloma or CLL. The treatment of multiple myeloma according to the present invention includes the treatment of smoldering myeloma. Smoldering myeloma is a precursor to active multiple myeloma. The treatment of smoldering myeloma may prevent or delay the progression to active multiple myeloma.

[0051] The cancers treated according to the present invention are present in human subjects (or patients). Neg LTK Pos The patient is diagnosed with cancer. Such a diagnosis can be made as described above. In a particular embodiment, the patient treated according to the present invention is an ALK Neg LTK Pos The patient is diagnosed with multiple myeloma. Alternatively, the patient is Neg LTK Pos The patient may have been diagnosed with CLL. Alternatively, the patient may have ALK Neg LTK Pos The patient may have been diagnosed with primary liver cancer. The patient may be of any age and may be male or female.

[0052] As mentioned above, cancers associated with protein hypersecretion (particularly multiple myeloma) are usually treated with proteasome inhibitors. To date, three proteasome inhibitors have been approved for the treatment of multiple myeloma: bortezomib (Formula VIII), carfilzomib (Formula IX), and ixazomib (Formula X). [ka] [ka] [ka]

[0053] A number of other proteasome inhibitors are known in the art, several of which are in clinical trials for the treatment of cancer, including marizomib, oprozomib, and MG132.

[0054] In certain embodiments, the cancer treated according to the present invention is resistant to treatment with a proteasome inhibitor. A cancer resistant to treatment with a proteasome inhibitor is a cancer whose growth is not inhibited by a proteasome inhibitor. That is, a cancer resistant to treatment with a proteasome inhibitor is a cancer that does not respond to treatment with a proteasome inhibitor, i.e., a cancer that continues to grow or progress despite treatment with a proteasome inhibitor.

[0055] Cancers that are resistant to treatment with proteasome inhibitors may be inherently resistant to treatment with proteasome inhibitors. As mentioned above, cancers characterized by protein hypersecretion are particularly sensitive to proteasome inhibitor therapy. This is because the increased synthesis of proteins in cancers characterized by protein hypersecretion results in increased levels of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. This is alleviated by the degradation of misfolded proteins by the proteasome. Proteasome inhibition inhibits the degradation of misfolded proteins, thus inhibiting the alleviation of ER stress, causing cell death. On the other hand, cancers that are not associated with protein hypersecretion may be less susceptible to proteasome inhibition and therefore resistant to treatment with proteasome inhibitors.

[0056] Some cancers are inherently resistant to proteasome inhibitor treatment due to mutations in the proteasome at the inhibitor binding site. For example, the A49T mutation in the β5 subunit of the human proteasome has been found to be associated with bortezomib resistance. Mechanisms by which cancers may become resistant to treatment with proteasome inhibitors are reviewed in Wallington-Beddoe et al., British Journal of Haematology 182(1): 11-28, 2018.

[0057] A cancer that is inherently resistant to treatment with a proteasome inhibitor is a cancer that does not respond at all to proteasome inhibitor treatment, i.e., a cancer in which the cancer does not respond to the treatment and continues to grow and / or progress when a proteasome inhibitor is first administered to a patient.

[0058] Alternatively, a cancer that is resistant to treatment with a proteasome inhibitor may have acquired resistance to a proteasome inhibitor. A cancer that has acquired resistance to a proteasome inhibitor is one that initially responded to treatment with a proteasome inhibitor, but has since lost its response to this treatment. This loss of response to proteasome inhibitor therapy may result in relapse or recurrence of the cancer in the treated patient. Cancers may acquire resistance to proteasome inhibitors by one or more of the mechanisms discussed above and in Wallington-Beddoe et al. (see above), particularly the development of mutations in the proteasome that prevent inhibitor binding.

[0059] A cancer that is resistant to treatment with a proteasome inhibitor may be resistant to treatment with one or more proteasome inhibitors. It is known that different proteasome inhibitors have different binding properties to the proteasome, and thus a cancer may be resistant to one proteasome inhibitor but sensitive to treatment with another proteasome inhibitor. In a particular embodiment, the cancer is resistant to treatment with bortezomib. In another embodiment, the cancer is resistant to treatment with carfilzomib. In another embodiment, the cancer is resistant to treatment with ixazomib. The cancer may be resistant to treatment with all proteasome inhibitors, particularly all proteasome inhibitors approved for cancer treatment. The cancer that is resistant to treatment with a proteasome inhibitor may be multiple myeloma, particularly multiple myeloma that is resistant to bortezomib treatment.

[0060] "Plasma cell mediated disease" refers to a disease or condition in which dysregulated plasma cells or antibodies produced by plasma cells are detrimental to health. That is, any disease or condition caused or contributed to by plasma cell activity is a plasma cell mediated disease. In plasma cell mediated diseases, antibodies produced by plasma cells cause tissue damage in patients. Such diseases are often associated with hypersecretion of antibodies from dysregulated plasma cells. The present invention provides linear inhibitors of ALK for use in treating plasma cell mediated diseases characterized by cells that are ALK negative and LTK positive. In this regard, ALK Neg And LTK Pos The cells which are are the plasma cells which mediate the disease.

[0061] Examples of plasma cell mediated diseases include systemic lupus erythematosus, ITP, and many autoimmune diseases such as graft-versus-host disease (GVHD). While GVHD is sometimes considered to be primarily a T cell mediated disease, it can also have a B cell component that is important in chronic rejection, where dysregulated plasma cells produce autoantibodies that contribute significantly to delayed graft rejection and disease effects. Thus, GVHD is considered herein to be a plasma cell mediated disease.

[0062] As defined herein, "plasma cells" refers to all types of cells of the plasma cell lineage, i.e., mature plasma cells and their precursors. As defined herein, plasma cells are defined as terminally differentiated B cells, including antibody-secreting plasmablasts and other antibody-secreting B cell blasts. Thus, "plasma cells" include mature plasma cells and short-lived plasmablasts. As known to those skilled in the art, activated memory B cell blasts may express CD20, which is associated with CD38. Hi CD138 Neg Plasma cells may be lost to plasmablasts, which are CD38 Hi CD138 Pos CD19 + or CD38 Hi CD138 Pos CD19 Neg It may be a terminally differentiated plasma cell.

[0063] Thus, in accordance with the present invention, linear ALK inhibitors may be used in autoimmune diseases, particularly for the treatment of autoimmune diseases characterized by the production of autoantibodies. Examples of autoimmune diseases treatable in accordance with the present invention include lupus and ITP. The present invention also provides for the treatment of GVHD using linear ALK inhibitors. As discussed above, non-cancerous plasma cells (e.g., plasmablasts and mature plasma cells) express ALK Neg LTK Pos Any plasma cell mediated disease (such as an autoimmune disease or GVHD) that is characterized by having an ALK phenotype and is therefore treated according to the present invention may be Neg LTK PosThe ALK and LTK expression is mediated by plasma cells that are ALK and LTK positive, respectively, although the ALK and LTK phenotypes of the plasma cells may be determined experimentally as described above.

[0064] As mentioned above, since LTK and ALK are expressed in all mammals, the present invention provides linear ALK inhibitors for treating autoimmune diseases in any mammalian subject, including human and non-human mammals, and in particular ALK inhibitors. Neg LTK Pos Linear ALK inhibitors are provided for treating autoimmune diseases characterized by the production of autoantibodies by plasma cells. Examples of non-human mammals that may be treated according to the present invention include dogs (canines), cats (felines), and horses (equines).

[0065] As mentioned above, linear ALK inhibitors are known to inhibit LTK.Therefore, the ALK inhibitor used in the treatment of cancer or plasma cell-mediated disease according to the present invention can be any known ALK inhibitor, as long as the inhibitor also has activity against LTK.Thus, in various aspects of the present invention as defined and described herein, said ALK inhibitor can also be defined as an ALK inhibitor that can inhibit LTK, or can specifically inhibit the activity of LTK.

[0066] In one embodiment, the ALK inhibitor has the general structure set forth in formula XI below: [ka] In the ALK inhibitor of formula XI, R1 is CH3 or iPr, R2 is H or CH3; [ka]

[0067] The ALK inhibitor used according to the present invention may in particular be ceritinib (formula III), which is also commercially available under the trade name Zykadia (e.g. from Cayman Chemical, USA, item no. 19374).

[0068] The standard dosing regimen for ceritinib in the treatment of NSCLC is a once-daily oral dose of 450 mg. In certain embodiments of the invention, the treatment with ceritinib comprises administering a once-daily dose of about 450 mg ceritinib, preferably a once-daily dose of 450 mg ceritinib. In another embodiment, the treatment comprises administering a once-daily dose of about 300 mg ceritinib or about 150 mg ceritinib, preferably a once-daily dose of 300 mg ceritinib or 150 mg ceritinib. In certain embodiments, the treatment comprises administering a once-daily dose of 100-400 mg ceritinib, for example, a once-daily dose of 200-400 mg, 100-300 mg, 200-300 mg, or 300-400 mg ceritinib. In another embodiment, the treatment comprises administering a once daily dose of 400-600 mg of ceritinib, e.g., a once daily dose of 400-500 mg, or a once daily dose of 500-600 mg of ceritinib. In a particular embodiment, the present invention relates to a treatment for ALK deficiency comprising administering a once daily dose of 400-500 mg of ceritinib, preferably a once daily dose of 450 mg of ceritinib. Neg LTK Pos The present invention provides a method for the treatment of multiple myeloma, comprising administering ceritinib orally to a patient having the disease.

[0069] In a specific embodiment, the invention provides ceritinib for use in the treatment of ALK-negative and LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 450 mg of ceritinib daily, said dose being administered orally.

[0070] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative and LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 300 mg of ceritinib daily, said dose being administered orally.

[0071] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative and LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 150 mg of ceritinib daily, said dose being administered orally.

[0072] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative, LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 500 to 600 mg of ceritinib per day, said dose being administered orally.

[0073] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative, LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 400 to 500 mg of ceritinib per day, said dose being administered orally.

[0074] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative, LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 300 to 400 mg of ceritinib per day, said dose being administered orally.

[0075] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative, LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 200 to 300 mg of ceritinib per day, said dose being administered orally.

[0076] In another embodiment, the invention provides ceritinib for use in the treatment of ALK-negative, LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 100 to 200 mg of ceritinib per day, said dose being administered orally.

[0077] In another embodiment there is provided ceritinib for use in the treatment of LTK positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 300 to 750 mg of ceritinib per day, said dose being administered orally.

[0078] In another embodiment, brigatinib is provided for use in treating LTK-positive multiple myeloma in a human subject, said treatment comprising administration of a dose comprising 50 to 200 mg of brigatinib per day, said dose being administered orally.

[0079] In another embodiment, a method of treating cancer in a patient diagnosed with multiple myeloma, chronic lymphocytic leukemia, or liver cancer is provided, said method comprising the steps of: obtaining a sample comprising cancer cells from said patient; subjecting the cancer cells to LTK expression analysis; and If the cancer cells express LTK, administering to the patient an effective amount of a linear ALK inhibitor.

[0080] In another embodiment, a method of treating cancer in a patient diagnosed with multiple myeloma, chronic lymphocytic leukemia, or liver cancer is provided, said method comprising the steps of: obtaining a sample comprising cancer cells from said patient; subjecting the cancer cells to LTK expression analysis; and If the cancer cells express LTK, administering to the patient an effective amount of an ALK inhibitor selected from ceritinib, brigatinib, crizotinib, ensartinib, and entrectinib.

[0081] In another embodiment, a method of treating an autoimmune disease in a patient diagnosed with lupus or immune thrombocytopenia is provided, the method comprising the steps of: obtaining a sample comprising plasma cells from said patient; subjecting the plasma cells to LTK expression analysis; and If the plasma cells express LTK, administering to the patient an effective amount of a linear ALK inhibitor.

[0082] In another embodiment, a method of treating an autoimmune disease in a patient diagnosed with lupus or immune thrombocytopenia is provided, the method comprising the steps of: obtaining a sample comprising plasma cells from said patient; subjecting the plasma cells to LTK expression analysis; and If the plasma cells express LTK, administering to the patient an effective amount of an ALK inhibitor selected from ceritinib, brigatinib, crizotinib, ensartinib, and entrectinib.

[0083] In another embodiment, a method of treating graft-versus-host disease in a patient is provided, the method comprising the steps of: obtaining a sample comprising plasma cells from said patient; subjecting the plasma cells to LTK expression analysis; and If the plasma cells express LTK, administering to the patient an effective amount of a linear ALK inhibitor.

[0084] In another embodiment, a method of treating graft-versus-host disease in a patient is provided, the method comprising the steps of: obtaining a sample comprising plasma cells from said patient; subjecting the plasma cells to LTK expression analysis; and If the plasma cells express LTK, administering to the patient an effective amount of an ALK inhibitor selected from ceritinib, brigatinib, crizotinib, ensartinib, and entrectinib.

[0085] Ceritinib may be administered in any suitable form.Various crystalline forms of Ceritinib are known, and are described in detail in WO2012 / 082972 and WO2016 / 098070.Any such crystalline form may be used according to the present invention.Preferably, Ceritinib is formulated and administered in capsule form.

[0086] In another embodiment, the ALK inhibitor used according to the present invention is brigatinib (Formula IV). Brigatinib is available under the trade name Alunbrig and is orally administered to patients according to a dosing regimen of 90 mg once a day for 7 days, followed by 180 mg once a day. In one embodiment of the present invention, brigatinib is administered according to this dosing regimen. In another embodiment, brigatinib is orally administered according to a dosing regimen of 60 mg once a day for 7 days, followed by 120 mg once a day, or 90 mg once a day. In another embodiment, brigatinib is administered at an oral dosage of 60 mg once a day. Preferably, brigatinib is formulated and administered in tablet form.

[0087] In another embodiment, the ALK inhibitor used according to the invention is crizotinib (Formula II). Crizotinib (brand name Xalkori) is typically administered to patients according to a dosing regimen of 250 mg taken orally twice a day. In one embodiment of the invention, crizotinib is administered according to this dosing regimen. In another embodiment, 200 mg of crizotinib is administered orally twice a day. In another embodiment, crizotinib is administered at an oral dose of 250 mg once a day. Preferably, crizotinib is formulated and administered in capsule form.

[0088] In another embodiment, the ALK inhibitor used in accordance with the present invention is ensartinib (Formula V). Ensartinib may be administered orally at a dosage of 225 mg once daily.

[0089] In another embodiment, the ALK inhibitor used according to the present invention is alectinib (Formula VI). Alectinib (trade name Alecensa) is typically administered to patients according to a dosing regimen of 600 mg taken orally twice a day. In one embodiment of the present invention, alectinib is administered according to this dosing regimen. In another embodiment, 450 mg of alectinib is administered orally twice a day. In another embodiment, 300 mg of alectinib is administered orally twice a day. Preferably, alectinib is formulated and administered in capsule form.

[0090] In another embodiment, the ALK inhibitor used according to the present invention is entrectinib (Formula VII). Entrectinib (trade name Rozlytrek) is typically administered to patients according to a dosing regimen of 600 mg taken orally once a day. In one embodiment of the present invention, entrectinib is administered according to this dosing regimen. In another embodiment, 500 mg of entrectinib is administered orally once a day. In another embodiment, 400 mg of entrectinib is administered orally once a day. In another embodiment, 300 mg of entrectinib is administered orally once a day. In another embodiment, 200 mg of entrectinib is administered orally once a day. Preferably, entrectinib is formulated and administered in capsule form.

[0091] Alternatively, any one of the linear ALK inhibitors disclosed in WO 2008 / 073687, WO 2009 / 126514, or WO 2010 / 002655 (all of which are incorporated by reference herein) may be used in the present invention.

[0092] For each agent, a suitable dosing schedule may be selected by a skilled physician, and may be selected based, for example, on clinical studies and the patient's response to the drug. ALK inhibitors are currently known for oral administration, but alternative routes of administration may be used, as appropriate, for example intravenous administration. The ALK inhibitors for use according to the invention are preferably administered in the form of a pharmaceutical composition. Suitable pharmaceutical compositions include solutions or syrups, solid compositions such as powders, granules, tablets, or capsules, as well as creams and ointments. Preferably, each ALK inhibitor, as defined above, inhibits ALK Pos It will be administered in a form approved for use in the treatment of NSCLC.

[0093] Pharmaceutically acceptable diluents, carriers, and excipients used in such compositions are well known in the art.For example, suitable excipients include lactose, sodium starch glycolate, corn starch or its derivatives, stearic acid or its salts, vegetable oils, waxes, fats, and polyols.Suitable carriers or diluents include carboxymethylcellulose (CMC), methylcellulose, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), dextrose, trehalose, liposomes, polyvinyl alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, silica, gelatin, oils and fats, alcohols, surfactants, and emulsifiers such as polysorbates.Stabilizers, wetting agents, emulsifiers, sweeteners, and the like may be used.Stabilizers, wetting agents, emulsifiers, sweeteners, and the like may also be used.

[0094] In preferred embodiments, the ALK inhibitors used in accordance with the present invention are formulated with excipients or the like that have been previously approved for use. Such information can be found in the regulatory approvals for these drugs issued by the FDA or EMA.

[0095] The term "treatment" as used herein includes both curative treatment (or treatment intended to be curative) and palliative treatment (i.e., treatment that merely limits, relieves or ameliorates the symptoms of cancer or plasma cell-mediated disease).

[0096] In one embodiment, the ALK inhibitor used according to the invention may be used as a single therapeutic agent, for example for the treatment of cancer or plasma cell mediated disorders. According to the invention, multiple ALK inhibitors may be used sequentially. This means that: a first ALK inhibitor is used to treat a patient's cancer, and then discontinued (e.g. because the patient responds poorly to the drug, or the cancer becomes resistant to it, etc.); after discontinuing the administration of the first ALK inhibitor, a second (different) ALK inhibitor is administered to the patient, which is also discontinued and a third different ALK inhibitor is administered (and so on). A similar policy may be applied, if necessary, to the treatment of plasma cell mediated disorders.

[0097] In another embodiment, the ALK inhibitor used according to the present invention may be part of a combination therapy. For example, in cancer treatment, the ALK inhibitor may be used together with at least one further anti-neoplastic drug or anti-neoplastic therapy. The further anti-neoplastic drug / therapy is not an ALK inhibitor. The administration of the ALK inhibitor may be combined with, for example, a conventional chemotherapy drug, a radiation therapy, a hormonal therapy or an immunotherapy, or a second targeted / high precision small molecule therapy. In the treatment of plasma cell mediated diseases according to the present invention, the ALK inhibitor may be used in combination with a suitable second therapeutic molecule. For example, in the treatment of autoimmune diseases, the ALK inhibitor may be used in combination with a drug used in the treatment of autoimmune diseases, such as an immunosuppressant, for example, a nonsteroidal anti-inflammatory drug (NSAID) or a corticosteroid. According to the present invention, ALK Neg LTK PosIn the treatment of multiple myeloma, ALK inhibitors may be used in particular in combination with proteasome inhibitors. The ALK inhibitor for use in the present invention may be used in combination with any one of the above proteasome inhibitors. For example, ceritinib may be used in combination with bortezomib, carfilzomib, or ixazomib.

[0098] The invention will be further understood by reference to the following non-limiting examples and drawings. [Brief description of the drawings]

[0099] [Figure 1] Figure 1: LTK knockdown causes endoplasmic reticulum stress. This figure shows the effect of LTK knockdown on endoplasmic reticulum stress caused by protein hypersecretion. HeLa cells encoding inducible IgM (heavy and light chains) were transfected with control or LTK siRNA. After 72 hours, cells were treated with mifepristone to induce IgM expression, then lysed (at the indicated times post-induction) and immunoblotted for the indicated proteins. [Diagram 2]Figure 2: LTK enables cells to cope with high secretory loads. A: L363 cells were treated with 5 μM crizotinib and the ratio of unspliced ​​to spliced ​​XBP1 expression and ATF4 expression were measured 4, 8, and 12 h after treatment. XBP1 splicing and ATF4 accumulation are signs of endoplasmic reticulum stress induction. B: Bortezomib-resistant L363aBTZ cells were treated with 5 μM crizotinib and the ratio of unspliced ​​to spliced ​​XBP1 expression and ATF4 expression were measured 4, 8, and 12 h after treatment. C: HeLa cells expressing inducible IgM (heavy and light chains) were treated with mifepristone to induce IgM expression for 2 weeks. Cells were then treated with 1 μM crizotinib for 24 hours, then lysed and immunoblotted against spliced ​​XBP1 to measure ER stress. + and - indicate cells treated with or without mifepristone, respectively. Induced cells are considered hypersecretory, whereas non-induced cells have normal levels of secretion. Numbers above the blots indicate densitometric quantification ± SD from three independent experiments. However, hypersecretory cells exhibit high levels of ER stress upon treatment with crizotinib. D: Comparison of LTK mRNA expression by quantitative PCR between HeLa cells not overexpressing IgM and cells overexpressing IgM for a long period (14 days). [Diagram 3]Figure 3: ALKNegLTKPos cells are sensitive to crizotinib treatment and undergo apoptosis. This figure shows that inhibition of LTK in hypersecretory cells reduces their viability. A: HeLa cells expressing inducible IgM (heavy and light chains) were treated with mifepristone to induce IgM expression for 2 weeks. Cells were then treated with 1 μM crizotinib for 24 h, then lysed and immunoblotted for caspase 3. The top blot shows uncleaved caspase 3 and the bottom blot shows cleaved caspase 3. Cleavage of caspase 3 indicates apoptosis induction, + and - indicate cells with or without mifepristone treatment, respectively. B: Concentration-response curves for the effect of crizotinib treatment (24 h) on the viability of three different myeloma cell lines. Results are the average of three independent experiments. C: LTK and ALK mRNA expression levels were measured by qPCR in the indicated myeloma cell lines. [Figure 4] Figure 4: ALKNegLTKPos cell lines are sensitive to ALK inhibitor treatment. This figure shows concentration-response curves for the effect of ceritinib, alectinib, ensartinib, lorlatinib, entrectinib, and crizotinib (24 h treatment) on the viability of myeloma cell lines L363 and AMO-1, and their bortezomib-resistant clones (L363-BTZ and AMO-1-BTZ). Results are the average of three independent experiments. Note the concentration-dependent decrease in myeloma cell viability. [Diagram 5]Figure 5: LTK regulates secretion and linear ALK inhibitors reduce secretion. This figure shows the effect of LTK inhibition by ceritinib on protein secretion. A: HeLa cells expressing flag-tagged LTK were treated with 1 μM crizotinib or ceritinib for 30 min. Cells were then lysed and immunoblotted with phosphorylated LTK, which was used as a surrogate for the active form of LTK. The lower blot shows the levels of total LTK in the cells. B: HeLa cells were treated with 1 μM ceritinib for 30 min, followed by fixation and staining for Sec31A to label ERES. Cells were imaged by confocal microscopy and ERES were counted using ImageJ. C: HeLa cells stably expressing mCherry-tagged mannosidase II RUSH reporter. Cells were treated with biotin to release the reporter from the ER. After 20 min, when most of the reporter had arrived at the Golgi, cells were fixed and immunostained for GM130 to label the Golgi apparatus. Images show a representative example of cells 20 min after biotin addition, where ceritinib-treated cells have less reporter expression in the Golgi region. The graph on the right shows quantification of the ratio of reporter fluorescence (red) in the Golgi (green) region. D: IgA-secreting AMO-1 cells were treated with the indicated doses of ceritinib for 3 h, and the amount of IgA in cell supernatants was measured by ELISA. [Figure 6] Figure 6: Multiple myeloma cells from patients are sensitive to crizotinib treatment. This figure shows a dose-response curve for the effect of crizotinib treatment (72 hours) on the viability of purified CD138+ multiple myeloma cells isolated from seven patients as indicated. Viability was measured by CellTiterGlo. [Figure 7]Figure 7: LTK and ALK expression in multiple myeloma cells from myeloma patients. A and B show omic data from the CoMMpass study, IA-13 build. Data included 767 multiple myeloma samples (www.themmrf.org) from the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (https: / / research.themmrf.org). RNA-seq data are expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). A: RNA-seq data from multiple myeloma cells from 767 patients from the CoMMpass study. Expression of LTK and secretory pathway genes such as ERGIC-53 (LMAN1), VIP36 (LMAN2), SURF4, BiP (HSP5A), ALK genes, and negative control IL-2 are shown. IL-2 is not expressed in multiple myeloma cells (expression is below the cutoff value (dotted line) of FPKM=1). The violin plot includes the median (dashed line) and quartiles (dotted line). B: Dot plot showing LTK expression versus ALK expression in 767 patients. The dotted line indicates the expression cutoff value (FPKM=1) for LTK and ALK. [Figure 8]Figure 8: Xenografted human multiple myeloma cells in NSG mice are sensitive to crizotinib. In this figure, immune-compromised NSG mice were injected intrafemorally with human L363-BTZ (bortezomib-resistant) myeloma cells. The cells carried luciferase to monitor disease progression in vivo. Starting 10 days later, mice were treated with bortezomib intravenously twice for 7 days and crizotinib orally daily for 7 days. The images on the left show examples of tumor burden in the different groups on days 4 and 7 of treatment (days 14 and 17 after tumor inoculation), and the graphs on the right show the average luciferase signal for each mouse in each cohort (n=4) normalized to day 0 of treatment (day 10 after tumor inoculation). Statistical differences between groups were analyzed by two-way ANOVA, and statistical differences between time points were analyzed by one-way ANOVA with Tukey post-test. [Figure 9] Figure 9: LTK and ALK expression in chronic lymphocytic leukemia (CLL) cells. Gene expression data from 107 patient CLL cell samples were analyzed for LTK and ALK expression. As shown, 94 of 107 patients (88%) of the CLL samples expressed LTK above the cutoff value (dotted line), while no samples expressed ALK (histogram on the right). [Figure 10]Figure 10: CLL cells from patients are sensitive to the linear ALK inhibitor crizotinib. This figure shows the response of activated and proliferating CLL blasts in response to a linear ALK inhibitor. Left: Dose-response curves of CLL cells from 21 patients. Center: Violin diagram showing 17 samples sensitive to crizotinib with IC50 values. These show supersensitive (CLL cells from 3 patients), highly sensitive (3 patients), and sensitive (11 patients). Right: Drug sensitivity scores (DSS values, Yadav et al., Scientific Reports 4: 5193,2014) integrate multiple dose-response relationships in high-throughput compound testing studies. The DSS of the CLL samples shows that one-third of patients have CLL cells that are highly sensitive to crizotinib. [Figure 11] Figure 11: CLL cells from IgVHUnmut patients are sensitive to crizotinib. The CLL samples from Figure 10 were classified with respect to the mutation status of the immunoglobulin variable heavy chain (IgVH) gene. Left: Four out of six patient samples with IgVHUnmut CLL are highly sensitive to crizotinib. None of the IgVHUnmut samples showed low sensitivity. Right: Three out of 15 IgVHMut CLL samples are highly sensitive to crizotinib. One of these was found to be marginally mutated (CLL168 with 97.9% homology). Four out of 15 IgVHMut samples showed low sensitivity. [Figure 12] Figure 12: CLL cells from patients are sensitive to ALK inhibitor treatment. CLL cells from 12 patients were examined for response to ALK inhibitor treatment with brigatinib, ceritinib, ensartinib, entrectinib, and lorlatinib. Drug sensitivity scores (DSS values, Yadav et al., Scientific Reports 4: 5193,2014) are shown. The cut-off line is set at DSS=10 (dotted line). [Figure 13]Figure 13: LTK and ALK expression in B cell differentiation. This figure analyzes the expression of ALK and LTK in subsets of B cells in humans. A: For B cell subsets, the expression signal is shown: LTK expression (left plot) and ALK expression (right). LTK is expressed above the cutoff value in memory cells, plasmablasts, and plasma cells. ALK is not expressed in B cell subsets. B: RNA-seq data was downloaded from deposited RNA-seq and flow cytometry data for 29 immune cell types within the peripheral blood mononuclear cell (PBMC) fraction of healthy donors. The data showed that naive B cells had low expression of LTK (18 out of 105 positive) and were negative for ALK. Memory B cells and plasma cells were positive for LTK and all negative for ALK. Memory B cells and plasma cells were ALKNegLTKPos. [Figure 14] Figure 14: In vitro generated plasma cells were sensitive to crizotinib. CD19+ B cells from five blood bank donors were stimulated for three days with adherent mouse L cells expressing CD40L, BAFF, and April to generate B cell blasts (plasmablasts / cell). Plasmablasts / cells were examined for sensitivity to crizotinib for 72 hours. Left: Relative cell viability in drug sensitivity profile of five normal plasmablasts / cells treated with crizotinib. Right: IC50 of five normal plasmablasts / cells treated with crizotinib. [Figure 15]Figure 15: In vitro generated plasma cells are sensitive to the linear ALK inhibitors Crizotinib and Ceritinib. Negatively selected normal B cells from blood bank donors were stimulated with sCD40L, IL-21, and IL-4 for 5 days. This protocol allows the generation of 10-20% terminally differentiated plasma cells expressing IRF4 and BLIMP1. Starting on days 5-7, cells were exposed to dose-tied crizotinib in A or dose-tied ceritinib in B. In A and B, IRF4 vs. BLIMP1 in gated CD19+ B cells are shown. Double positive IRF4 and BLIMP1 cells represent end-stage plasma cells. Such plasma cells were gated (blue oval region) and the percentage of plasma cells is shown. Conditions without inhibitor (0) and with linear ALK inhibitors (crizotinib, A; ceritinib, B) at concentrations of 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM are shown. Panel C shows the calculation of percent viability of plasma cells in response to the indicated doses of crizotinib and ceritinib. [Figure 16] Figure 16: Plasma cells are sensitive to crizotinib in vivo and reduce Ig secretion in vitro. Luciferase+L363wt plasma cell line was injected intra-femorally into immune-compromised NSG mice. Ten days later, mice were treated with crizotinib daily for seven days (50 mg / kg per day, orally). Left: Plasma cell line is killed by crizotinib in xenografted mice. Mice were imaged with an IVIS optical imager on days 10, 14, and 17. Ventral aspects of three of four mice are shown. Mice were given vehicle (top) or crizotinib (bottom). Crizotinib significantly reduced signal from plasma cells in three of four mice. Right: Ig secretion is reduced by ceritinib. The plasma cell line AMO-1 was washed and loaded into the wells (106 cells / well) and subjected to a titration of ceritinib for 3 hours. Supernatants were analyzed for IgA secreted by AMO-1 cells using a Human IgA ELISA Kit (Abcam, Cambridge, UK). [Figure 17]Figure 17: Liver hepatocellular carcinoma is ALKNegLTKPos and sensitized to apoptosis by crizotinib. RNA-seq data from 365 hepatocellular carcinomas (HCC) were generated from the Cancer Genome Atlas (TCGA) and reported as FPKM (number of Fragments Per Kilobase of exon per Million reads). 365 patient samples from hepatocellular carcinoma were examined for expression of LTK and ALK. In A, the plot on the left shows LTK vs. ALK, with the cutoff value shown as a dotted line. All patients have samples that are ALKNeg. A quarter of the samples are LTKPos and therefore ALKNegLTKPos. In B, hepatocellular carcinoma cells (HepG2) were treated with DMSO (untreated) or increasing concentrations of doxorubicin (1, 2, and 5 μM) for 24 hours and examined for apoptosis by immunoblotting for cleaved caspase 3. Doxorubicin at 5 μM only slightly induced cell death. Cells were also treated with the same concentration of doxorubicin for 24 h in the presence of 1 μM crizotinib. The presence of crizotinib sensitized HepG2 cells to the apoptosis-inducing drug doxorubicin, indicating that the provision of a linear ALK inhibitor sensitized HCC cells to apoptosis. EXAMPLES

[0100] Myeloma Experiments, Materials, and Methods Processing of patient samples and primary multiple myeloma cell samples Patients with multiple myeloma were recruited from the Oslo Myeloma Centre, Oslo University Hospital. The study was approved by the regional Committee for Medical and Health Research Ethics of South-East Norway (REC#2016 / 947 and 2012 / 174). Bone marrow aspirates were obtained from patients with multiple myeloma after signing informed consent in accordance with the Declaration of Helsinki. Samples from seven multiple myeloma patients were included. Patients were treated after second relapse (patient no. 1701), third relapse (2101, 2021, 1802, 9041, 2504), or fourth relapse (2201). Patients received a proteasome inhibitor (bortezomib or carfilzomib) in at least one line of treatment. Bone marrow mononuclear cells (BMMCs) were prepared from bone marrow aspirates of patients by Lymphoprep density gradient centrifugation. After CD8 depletion with Dynabeads (Life Technologies), BMMCs were stimulated by expanding Th cells in the presence of Human T-Activator CD3 / CD28 Dynabeads (Life Technologies) and 100 U / ml human interleukin-2 (hIL-2, Roche, Mannheim, Germany). After 48 h, BMMCs were enriched for CD138+ and multiple myeloma plasma cells were isolated using MACS CD138+ microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0101] Drug therapy and cell viability analysis (multiple myeloma patient cells) CD138+ multiple myeloma cells (5,000–10,000 cells / well) from the activation assay were tested in 384-well plates for their response to crizotinib. Six concentrations of crizotinib were tested in 10-fold dilutions covering a concentration range of 0.1–10,000 nM (plates dispensed with an Echo acoustic dispenser (LabCyte, San Jose, CA, USA)). The plates were incubated in a humidified environment at 37°C and 5% CO2. After 72 hours, cell viability was determined using a CellTiterGlo (Promega, Madison, WY, USA) ATP assay according to the manufacturer's instructions and using an Envision Xcite plate reader (Perkin Elmer, Shelton, CT, USA). Relative percentage cell viability was calculated by normalizing luminescence units for each well to negative (DMSO 0.1%) and positive (100 μM BzCl) controls and curve fitting was performed to determine IC 50 Got the value.

[0102] Cell culture and transfection HeLa cells were cultured in DMEM (GIBCO) supplemented with 10% FCS and 1% penicillin / streptomycin (GIBCO). For plasmid overexpression, cells were transfected using Fugene6 or TransIT-LT1 (Mirus). For knockdown experiments, cells were reverse transfected with 10 nM siRNA (final concentration) using HiPerfect (Qiagen) according to the manufacturer's instructions.

[0103] Cell lysis and immunoblotting Cells were washed twice with PBS and harvested in lysis buffer (50 mM Tris-HCl, pH 7.4; 1 mM EDTA, 100 mM NaCl, 0.1% SDS, and 1% NP-40) supplemented with protease and phosphatase inhibitors (Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-free). Lysates were incubated on ice for 10 min, followed by a wash centrifugation at 20,000xg for 10 min at 4°C. Supernatants were transferred to clean tubes and reducing loading buffer was added. Lysates were subjected to SDS-PAGE and transferred by semi-dry transfer onto nitrocellulose membranes. The membranes were blocked (in ROTI buffer (Roth) or 5% milk in PBS with 0.1% Tween) and probed with the appropriate primary antibodies. Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoblots were developed using chemiluminescence reagents (ECL clarity, BioRad) and imaged using a ChemiDoc (BioRad).

[0104] Multiple myeloma cell lines, cell culture, and viability analysis The cell lines used were human multiple myeloma cell lines L363 (DSMZ catalog number ACC49), AMO-1 (DSMZ catalog number ACC538), and ARH-77 (ATCC CRL-1621). Multiple myeloma cell lines were maintained in RPMI-1640 culture medium (Sigma-Aldrich, Bax, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μg / ml streptomycin, and 100 U / ml penicillin / streptomycin (Sigma-Aldrich, Bax, Switzerland). Bortezomib-resistant cell lines were established and maintained from parental cell lines by continuous drug exposure for over 12 months (Soriano et al., Leukemia 30:2198-2207, 2016). The viability of the cells was determined after 24 hours post-treatment using the Cell Counting Kit-8 (CCK-8; MedChemExpress, NJ, USA) according to the manufacturer's instructions.

[0105] Gene expression analysis Omics data can be accessed through CoMMpass SM The data were downloaded from the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (https: / / research.themmrf.org) and included 767 multiple myeloma samples. These data were generated as part of the Multiple Myeloma Research Foundation Personalized Medicine Initiatives (https: / / research.themmrf.org). RNA-seq data were expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). FKPM<1 was considered negative.

[0106] RNA isolation and RT-PCR (s / uXBP1 and ATF4 induction) Total RNA was isolated from cell lines using Trizol (Ambion / Thermo Fisher Scientific, MA, USA) and Direct-zol RNA MiniPrep (Zymo Research, CA, USA). 500 ng of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems / Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Then, 10 ng of cDNA was used per qPCR reaction in the QuantStudio5 Real-Time PCR system (Applied Biosystems / Thermo Fisher Scientific, MA, USA) using 2xPowerUp SYBR Green Master Mix (Applied Biosystems / Thermo Fisher Scientific, MA, USA) and the following primers: Primers: spliced, unspliced, and total XBP1, as well as ATF4 (Oslowski & Urano, Methods in Enzymology 490: 71-92, 2011), and GAPDH as a housekeeping gene control.

[0107] ELISA 1x10 6 / ml IgA-secreting AMO-1 cells were seeded in fresh medium containing increasing doses of the compound of interest for 3 hours. The medium was then collected and diluted 20-fold, and the levels of IgA antibodies in the cell culture supernatants were measured using a Human IgA ELISA Kit (Abcam, UK, product no. ab137980) according to the manufacturer's instructions.

[0108] In vivo experiments NSG (NOD-scid IL2Rgamma) was purchased from Jackson Laboratory (No. 005557, California, USA).null ) mice were obtained and kept in individually ventilated cages with free access to food. 5 L363-BTZ_Luc+_TdTomato+ multiple myeloma cells (with luciferase vector #72486 obtained from Addgene for monitoring) were injected into the right thigh. After luciferin injection (150 mg / kg, obtained from BioVision, CA, USA), tumor growth was monitored twice during one week. Treatment was started 10 days after tumor inoculation with crizotinib (50 mg / kg, orally, daily), bortezomib (1 mg / kg, intravenous injection, twice in 7 days), or vehicle for 7 days. The study was performed according to the 3R principle. The experiment was approved by the Committee for Animal Experiments, St. Gallen, Switzerland (application number 30177). Bortezomib and crizotinib were purchased from MedChemExpress, NJ, USA.

[0109] CLL Experiments, Materials, and Methods Gene Expression Data Gene expression data for CLL cells were obtained by analysis of previous datasets available on the HYPERLINK "http: / / www.genomicscape.com / microarray" http: / / www.genomicscape.com / microarray platform. Data from CLL cells of 107 CLL patients were analyzed for expression of LTK and ALK genes. Data were normalized with Robust Multiarray Averaging (RMA) from the Herold, chronic lymphocytic leukemia, HGU133P (107 samples) dataset (Herold et al., Leukemia 25(10): 1639-45, 2011) from the Affymetrix Human Genome U133 Plus 2.0 Array.

[0110] Dose-response experiments on CLL samples CLL cells from patients were stimulated with adherent mouse L cells expressing CD40L, BAFF, and April for 24 hours. The L cells were then removed by immunomagnetic separation. Washed CLL cells were plated in a 384-well plate format (10,000 cells / well) and tested for crizotinib at five concentrations in 10-fold dilutions (plates dispensed with an Echo acoustic dispenser (LabCyte, San Jose, CA, USA)). The plates were incubated in a humidified environment at 37°C and 5% CO2. After 72 hours, cell viability was measured using a CellTiterGlo (Promega, Madison, WY, USA) ATP assay according to the manufacturer's instructions and using an Envision 2102 Multilabel reader (PerkinElmer, Shelton, CT, USA). The relative percentage of cell viability was calculated by normalizing the luminescence units for each well to the negative control (DMSO 0.1%) and positive control (100 μM BzCl) wells. Curve fitting was performed to determine the IC 50 Got the value. A quantitative score of differential drug susceptibility for each drug was calculated as a drug susceptibility score (DSS value, Yadav et al., Scientific Reports 4:5193, 2014). DSS values ​​were calculated for pooled multiple dose-response relationships in high-throughput compound studies as indicated.

[0111] Gene expression analysis of human B cells Gene expression data for human B cell differentiation was obtained by analysis of datasets available from the HYPERLINK "http: / / www.genomicscape.com / microarray" http: / / www.genomicscape.com / microarray platform. The GEP dataset was derived from human plasma cell differentiation normalized by MAS5 on the Affymetrix Human Genome U133 Plus 2.0 Array (Jourdan et al., Leukemia 28(8): 1647-56, 2014; Jourdan et al., Journal of Immunology 187(8): 3931-41, 2011; Jourdan et al., Blood 114(25): 5173-81, 2009; Caron et al., Journal of Immunology 182(12): 7595-602, 2009). Data from donors were analyzed for expression of LTK and ALK genes during human B lymphopoiesis.B cells were classified into the following subsets: 1. naive B cells (n=5); 2. centroblasts (n=4); 3. centroblasts (n=4); 4. memory B cells (n=5); 5. pre-plasmablasts (n=5); 6. plasmablasts (n=5); 7. early plasma cells (n=5); 8. bone marrow plasma cells (n=5). RNA-seq data were downloaded from the deposited data (Monaco et al., Cell Reports 26(6): 1627-40, 2019). Data were analyzed for 29 immune cell types within peripheral blood mononuclear cell (PBMC) fractions of healthy donors using RNA-seq and flow cytometry. Naive B cell data was from Schmiedel et al., Cell 175(6): 1701-15, 2018.

[0112] Drug sensitivity screening of activated B cell blasts / plasma cells CD19 from five blood bank donors +B cells were stimulated for 3 days with adherent mouse L cells expressing CD40L, BAFF, and April to generate B cell blasts (plasmablasts / cell). Washed plasmablasts / cells were dissociated and plated in a 384-well format (10,000 cells / well). For crizotinib, 10 3 Six concentrations were tested in 10-fold dilutions, covering a concentration range of ~10 μM. After 72 hours, cell viability was measured using CellTiterGlo. Relative percentage cell viability was calculated by normalizing luminescence units for each well to the negative (DMSO 0.1%) and positive (100 μM BzCl) controls, and curve fitting was performed to determine IC 50 obtained.

[0113] result LTK is a regulatory node in the proteostasis network Mathematical models suggested that LTK is a positive regulator of ER trafficking activated by the secretory stream. To experimentally confirm this prediction, we used HeLa cells inducibly expressing IgM (Bakunts et al., see above). Induction of IgM expression for 24 h led to a mild activation of the UPR, a marker of ER stress, as indicated by increased levels of XBP1s (Fig. 1). When LTK expression was suppressed, cells responded to induction of IgM expression with increased levels of ER stress (Fig. 1). This confirms that LTK is a regulatory node of the proteostasis network.

[0114] LTK helps multiple myeloma cells cope with an increased secretory burden Multiple myeloma is characterized by the secretion of excessive amounts of immunoglobulins. To determine whether LTK inhibition induces ER stress in multiple myeloma cell lines, L363 cells were treated with crizotinib, a compound known to inhibit LTK and affect its secretion (Centonze et al., supra). Crizotinib treatment induced ER stress as evidenced by increased splicing of XBP1 and increased induction of ATF4 (Figure 2A). Similar findings were made for L363-BTZ cells, which are resistant to bortezomib (Figure 2B). However, L363 cells and their bortezomib-resistant clones are positive for LTK but negative for ALK (see below), minimizing the possibility of off-target effects of crizotinib. To investigate whether LTK helps cells adapt to high secretory load, we used a system that allows direct comparison of the same cells with and without hypersecretion. Rather than using only short-term pulse induction of IgM production and secretion, IgM production was induced for 14 days to take into account potential cellular adaptation mechanisms to chronic overexpression of secretory proteins. HeLa cells induced to overexpress IgM for 14 days were compared to non-induced HeLa cells. When non-induced cells were treated with the LTK inhibitor crizotinib, there was no significant increase in ER stress (Figure 2C), whereas cells subjected to an increased secretory load for a prolonged period of time exhibited 2.5-fold higher levels of ER stress (Figure 2C). The high reliance of hypersecretory cells on LTK is consistent with the finding that hypersecretory cells increased LTK expression 4-fold (Figure 2D).

[0115] Targeting LTK reduces viability of multiple myeloma cells Based on the observation that LTK inhibition induces ER stress in hypersecretory cells, we investigated whether it also causes cell death. We investigated the effect of crizotinib on caspase 3 cleavage in HeLa cells with and without IgM hypersecretion. Nonsecretory cells showed no apoptotic response to crizotinib treatment (Figure 3A), consistent with the observation that these cells do not respond to ER stress. However, cells with chronically increased secretory load showed a marked increase in apoptosis upon crizotinib treatment (Figure 3A). Treatment of two multiple myeloma cell lines (L363 and ARH77) with crizotinib resulted in a clear decrease in cell viability at pharmacologic relevant drug concentrations. The same was found for bortezomib-resistant clones of L363 (Figure 3B). However, these cells were positive for LTK but negative for ALK (Figure 3C), as were multiple myeloma cells obtained from the patient (see below).

[0116] Similar to crizotinib, other linear ALK inhibitors, such as ceritinib, alectinib, ensartinib, and entrectinib, were also found to exhibit cytotoxic activity against multiple myeloma cells (Figure 4, left panel). The cyclic ALK inhibitor lorlatinib did not efficiently kill myeloma cells. 50 Values ​​(μM) obtained for ceritinib in the cell lines investigated: 1.03 in L363, 0.79 in L363-BTZ, 2.09 in AMO-1, and 2.77 in AMO-1-BTZ. The following IC 50 Values ​​(μM) obtained for crizotinib in the cell lines examined were: 3.323 in L363, 4.15 in L363-BTZ, 3.92 in AMO-1, and 6.477 in AMO-1-BTZ. The following IC 50Values ​​(μM) were obtained for alectinib in the cell lines examined: 6.12 in L363, 6.22 in L363-BTZ, 13.31 in AMO-1, and 17.6 in AMO-1-BTZ. Similar sensitivity to LTK inhibition was seen for the bortezomib-resistant versions of L363 and AMO-1 (Figure 4, right). This suggests that LTK acts on a proteasome-independent part of the protein homeostasis network and that proteasome inhibitor resistance in multiple myeloma can be overcome by inhibiting LTK. This can be achieved by using approved ALK inhibitor drugs.

[0117] Mechanistically, linear ALK inhibitors such as ceritinib inhibit LTK (Figure 5A), which inhibits ER-Golgi transport (Figures 5B, C) and, consequently, immunoglobulin secretion (Figure 5D).

[0118] The effect of crizotinib was then examined on primary myeloma cells obtained from relapsed patients undergoing third- to fifth-line therapy. CD8-depleted bone marrow-derived mononuclear cells were stimulated with anti-CD3 / CD28 beads in IL-2. This method stimulated CD4+ helper T cells (Th cells) that support multiple myeloma cell activation. After 48 hours, CD138+ multiple myeloma cells from each of the 15 patients were transferred to wells in the presence of dose-set crizotinib and concentration-response curves were calculated (Figure 6). LTK inhibition had a significant effect on multiple myeloma cells from all patients, with IC 50 In particular, analysis of a cohort of 767 patients with multiple myeloma revealed that the mean expression level of LTK mRNA was 8.18, whereas the mean expression level of ALK was 0.02 (Figure 7A), a cutoff value (10 0 The patients were divided into the following categories: Alk, found in 636 of 767 patients (83%); Neg LTK Pos found in 130 of 767 patients (17%), Neg LTK Negand Alk, found in only 1 of 767 patients (0.001%). Pos LTK Pos Myeloma cells that are LTK-dependent (Figure 7B). Because ALK inhibitors do not have ALK as a target, the killing of primary myeloma cells by crizotinib is likely a result of LTK inhibition.

[0119] The in vivo effect of crizotinib on multiple myeloma cells was investigated. For this purpose, bortezomib-resistant L363 cells modified to express luciferase were injected into the thighs of immune-compromised mice. The cells were modified to express luciferase to facilitate monitoring of tumor growth in vivo. This model takes into account both the special physiology of proteasome inhibitor-resistant multiple myeloma cells and the protective and drug resistance-promoting properties of the bone marrow microenvironment, which are important for the physiology of multiple myeloma in vivo. Treatment with crizotinib was started at a stage when tumors were easily detectable (day 10 after tumor inoculation), mimicking the clinical setting. Mice were also treated with bortezomib, which was expected to have no effect given the use of bortezomib-resistant L363 cells, but proteasome inhibitor resistance was confirmed. Mice were orally administered crizotinib (50 mg / kg) daily for 7 days. Bortezomib was administered intravenously (1 mg / kg) twice during the above period. Treatment with crizotinib reduced tumor burden compared to baseline, untreated controls, and bortezomib-treated mice (Figure 8), indicating that treatment with an LTK inhibitor can overcome proteasome inhibitor resistance in multiple myeloma.

[0120] Primary CLL cells are ALK Neg LTK Pos is For chronic lymphocytic leukemia (CLL), we examined the transcription of ALK and LTK, and 94 of 107 CLL cell samples (88%) were found to express LTK. Pos All 107 samples were ALK NegTherefore, the majority of CLL patients were ALK-positive. Neg LK Pos CLL is a cancer in which leukemic cells in the blood are inactive or in a pre-apoptotic state. Division of CLL cells occurs in specialized microenvironments in lymph nodes, spleen, or bone marrow, where dividing CLL blasts reside next to stimulator cells in a microenvironment (so-called pseudofollicles) that supports lymphocytic blastogenesis and mitosis. At this stage, CLL cells are hypersecretory, secreting monoclonal antibodies before undergoing cell division (Darwiche et al., supra).

[0121] Primary CLL cells are sensitive to linear ALK inhibitors We prepared microenvironmental factors important for CLL cell activation (Patten et al., Blood 111: 5173-5181, 2008; Bagnara et al., Blood 117: 5463-5472, 2011; Hall et al., Blood 105: 2007-2015, 2005; Os et al., Cell Reports 4(3): 566-577, 2013) and tested proliferating CLL blasts for sensitivity to linear ALK inhibitors. We found that 17 of 21 patient samples (81%) had CLL cells that were sensitive to the linear ALK inhibitor crizotinib (Figure 10). Of these, 11 had normal sensitivity (IC 50 <2 μM, and three were highly sensitive (IC 50 <2x10 -1 μM), and three were highly sensitive (IC 50 <10 -2 Further integration of multiple dose-response relationships from high-throughput compound testing revealed that drug sensitivity scores (DSS) indicated that one-third of patients had CLL cells that were highly sensitive to crizotinib.

[0122] Patients with poor prognosis often have CLL cells that are sensitive to linear ALK inhibitors CLL is divided into two subtypes depending on the mutational burden of the V region of the immunoglobulin variable heavy chain gene (IgVH), as detailed above. Subtypes without IgVH mutations have a poor prognosis, whereas those with IgVH mutations (<98% homology to germline) are seen in indolent CLL with a better prognosis. These results showed that the poor prognosis subtype had a high response rate and sensitivity to linear ALK inhibitors (Figure 11). This suggests that linear ALK inhibitors are particularly relevant for the treatment of the poor prognosis subtype without IgVH mutations. CLL cells from 12 new patients were examined for sensitivity to ALK inhibitors. As shown in Figure 12, nearly all patients had CLL cells that showed sensitivity to ceritinib, crizotinib, ensartinib, and entrectinib (using a cutoff value of DSS=10, the results are 11 / 12, 10 / 12, 11 / 12, and 12 / 12). Brigatinib inhibited CLL cells from 4 of 12 patients, and lorlatinib inhibited CLL cells from 0 of 12 patients.

[0123] Normal memory B cells, plasmablasts, and plasma cells express ALK Neg LTK Pos We next considered normal B cells and the B cell differentiation lineage, including naive mature B cells, germinal centroblasts, germinal centrocytes, memory B cells, pre-plasmablasts, plasmablasts, early plasma cells, and bone marrow plasma cells. Gene expression (array-based) and RNA sequencing showed that memory B cells, plasmablasts, and plasma cells all expressed LTK but not ALK (Figure 13). Memory B cells, plasmablasts, and plasma cells therefore expressed ALK. Neg LTK Pos It is.

[0124] Normal memory B cells, plasmablasts, and plasma cells are sensitive to linear ALK inhibitors Activated B cells became B cell blasts after 3 days. These giant cells secrete high levels of antibodies and are therefore hypersecretory. B cells were stimulated on day 3 and cultured with the linear ALK inhibitor for an additional 3 days. All samples were stimulated with crizotinib at IC between 1 and 1.5 μM. 50 The B cells had similar sensitivity to ALK inhibitors (crizotinib or ceritinib) (Figure 14). The results show that at least half of the blast-like B cells (memory B cell blasts, plasmablasts, and plasma cells) are killed during the 3-day treatment. To further examine the sensitivity of the cell subtypes, B cells were stimulated for 5 days to generate plasma cells. End-stage plasma cells typically express IRF4 and BLIMP1 (Figure 15) and secrete high levels of antibodies. Exposure to a linear ALK inhibitor (crizotinib or ceritinib) efficiently killed the highly sensitive plasma cells. The plasma cell response was evaluated by IC 50 were 0.8 μM (crizotinib) and <0.001 μM (ceritinib), indicating that plasma cells were highly sensitive to linear ALK inhibitors. Although there was a reduction in all other activated B cell subsets and plasmablasts, plasma cells were the most sensitive to linear ALK inhibitors and were also highly sensitive to ceritinib.

[0125] Crizotinib kills plasma cells in xenografted NSG mice ALK Neg LTK Pos Plasma cell lines were injected intrafemorally into NSG mice. Treatment of mice with crizotinib reduced the density of plasma cells (Figure 16, luciferase-dependent signal, in IVIS optical imaging), indicating that plasma cell lines can also be inhibited in vivo.

[0126] Ceritinib inhibits antibody secretion by plasma cells in vitro We examined whether plasma cell secretion was inhibited with a linear ALK inhibitor (Figure 16). A 3-hour exposure reduced the levels of immunoglobulin secreted from plasma cell lines, suggesting a blockade of endoplasmic reticulum-to-Golgi transport, as shown in Figure 5A-C.

[0127] Hepatocellular carcinoma (HCC) cells express ALK Neg LTK Pos and was sensitive to crizotinib RNA-seq data from 365 HCCs showed that all patients had ALK Neg Approximately one-quarter of the samples had HCC samples that were LTK Pos and therefore ALK Neg LTK Pos In an experiment in which the linear ALK inhibitor crizotinib synergized with the cell proliferation inhibitor doxorubicin, cells from the well-differentiated HCC cell line HepG were found to be sensitive to the linear ALK inhibitor crizotinib ( FIG. 17 ). Thus, crizotinib sensitized HepG HCC cells to apoptosis.

Claims

1. A pharmaceutical composition comprising an anaplastic lymphoma kinase (ALK) inhibitor for use in treating a subject suffering from (i) or (ii) of the following: (i) ALK-negative and leukocyte tyrosine kinase (LTK)-positive (ALK Neg LTK Pos ) cancer selected from multiple myeloma or chronic lymphocytic leukemia; (ii) a plasma cell-mediated disease mediated by ALK Neg LTK Pos cells, selected from an autoimmune disease or graft-versus-host disease (GVHD); said cancer or plasma cell mediated disease is characterized by hypersecretion of antibodies by said ALK Neg LTK Pos cancer or plasma cells; and The pharmaceutical composition, wherein the ALK inhibitor is selected from ceritinib, brigatinib, crizotinib, ensartinib, alectinib, and entrectinib.

2. The pharmaceutical composition of claim 1 , wherein the cancer is multiple myeloma.

3. The pharmaceutical composition according to claim 1 , wherein the cancer is chronic lymphocytic leukemia or liver cancer.

4. The pharmaceutical composition of claim 1 , wherein the cancer is resistant to treatment with a proteasome inhibitor.

5. The pharmaceutical composition of claim 4 , wherein the proteasome inhibitor is bortezomib.

6. The pharmaceutical composition of claim 1 , wherein the autoimmune disease is lupus or immune thrombocytopenia.

7. 7. The pharmaceutical composition of any of claims 1 to 6, wherein the treatment comprises daily administration of a dose containing 450 mg of ceritinib, the dose being administered orally.

8. 7. The pharmaceutical composition of any of claims 1 to 6, wherein said treatment comprises administration of a dose comprising 200 mg to 400 mg of ceritinib per day, said dose being administered orally.

9. 7. The pharmaceutical composition of any of claims 1 to 6, wherein the treatment is for multiple myeloma, the treatment comprising administration of a dose comprising 400 mg to 500 mg of ceritinib per day, the dose being administered orally.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the subject is a human.

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