Method of treatment of p53 WT tumors

Abstract

To provide combinations for use in treating p53 wild type (WT) tumors.SOLUTION: Provided is a combination comprising (a) a mouse double minute chromosome 2 (MDM2) inhibitor and (b) a Casein Kinase 1 alpha (CK1α) degrading agent and / or a mouse double minute chromosome 4 (MDM4) inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications and Support This application is filed on October 1, 2018, the contents of which are incorporated by reference in their entirety. Priority to and claims priority to U.S. Provisional Patent Application No. 62 / 752,382, filed on the 30th. Claim the benefits of.

[0002] This invention was made with Government support under the National Institutes of The grant was awarded by the Health Grant Number R01 CA063113 R01 CA1 The government has granted certain rights in this invention under the provisions of CA203655 and CA73023. have the right.

[0003] The present invention relates to a method for treating p53 wild-type (WT) tumors. The inhibitor of double minute chromosome 2 (MDM2) inhibits casein kinase 1 alpha (CK1α) A new therapeutic approach for p53WT tumors based on combination therapy with anti-p53 and / or MDM4 inhibitors Provide new therapies. [Background technology]

[0004] Merkel cell carcinoma (MCC) is an aggressive neuroendocrine cancer of the skin with an incidence rate of It has tripled in the last 20 years.[1,2] In 2008, Feng et al. Clonally integrated Merkel cell poliomyelitis in eight of the MCC tumors We discovered that MCV and MCPyV are the most common mycoviruses in Japan [3]. It contains an integrated copy of the . ) [4]. MCC tumor-associated truncated LTs are expressed as N-terminal L XCXE, C-terminal DNA binding that retains the RB-binding motif but is required for viral replication and the helicase domain is deleted [3]. Expression of MCV ST and truncated LT is , which can promote proliferation and transformation in several cell types, and their This is consistent with a carcinogenic role.[5]

[0005] The prototype polyomavirus, simian vacuolar virus 40 (SV40)LT, is associated with retinoblastoma Binds to the protein RB (RB1) and the cellular tumor antigen p53 (TP53) and inhibits the expression of these tumors. In contrast, MCV LT binds to RB but not to p5 3. Next-generation sequencing of MCC has revealed that virus-negative MCC is typical. reveal that they harbor p53 and RB mutations with a UV damage signature [7,8] In contrast, virus-positive MCCs usually contain wild-type RB and p53. There is no evidence of UV damage [7,8]. Considering the presence of live p53, the present inventors determined that MCV T antigen functionally inactivates p53 activity. I doubted it could be activated.

[0006] p53 is mutated in a wide range of cancers. Instead, wild-type p53 is involved in the transcription of MDM2, Ubiquitin ligase that targets p53 or functions upon overexpression of MDM4 (MDMX) Both MDM2 and MDM4 bind N-terminally to p53 and can be selectively inactivated [9,10]. MDM4 has a similar structure with a C-terminal RING domain and a C-terminal RING domain

[11] . Although it does not directly ubiquitinate MDM2, its RING domain mediates the recruitment of ubiquitin to MDM2.

[11] MDM4 also contains an autoinhibitory domain that reduces binding to p53.

[12] The MDM4 autoinhibitory interaction is related to the interaction with casein kinase 1 alpha (CK1α, This can be alleviated by the treatment with CSNK1A1.

[13] Summary of the Invention [Means for solving the problem]

[0007] The present invention provides an MDM2 inhibitor for use in treating a p53WT tumor in a subject. and a casein kinase 1 alpha (CK1α) degrader and / or an MDM4 inhibitor. Provide a new combination.

[0008] In the combination of the present invention, the MDM2 inhibitor is Nutlin-3, Idasanutlin (RG7388, RO5503781, Roche), RG7775(RO683992 1, Roche), RO5045337 (Roche), AMG232 (Amgen), DS3032 (DS3032b, Daiichi Sankyo), ALRN-6924 (Aileron), KRT-232 (Kartos), ATSP-7041, CGM0 97 (Novartis) and HDM201 (Novartis). Preferably, HDM201, i.e., (S)-5-(5-chloro-1-methyl-2-oxopropyl) So-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-( 2,4-Dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro- 1H-pyrrolo[3,4-d]imidazol-4-one.

[0009] In the combination according to the present invention, a casein kinase 1 alpha (CK1α) degrading agent and and / or MDM4 inhibitors include thalidomide, pomalidomide, lenalidomide and SC-24 -UR99 (Novartis), i.e. 4-(3-amino-1-(4-chloro-5-methyl) 6-(methylamino)pyridin-3-yl)-5-fluoro-1H-indazole 6-yl)naphthalene-1-ol, preferably lenalidomide (RS)-3-(7-amino-3-oxo-1H-), also known as REVLIMID isoindole-2-yl)piperidine-2,6-dione.

[0010] The present invention relates in particular to a compound having an MDM2 inhibitor HDM201 and a CK1α degrader lenalidomide. Such a combination is provided.

[0011] The combination according to the invention may include a further anti-cancer agent.

[0012] Further anticancer agents according to the present invention include FLT3 inhibitors (e.g., gilterinib, quizartinib, midostaurin), BCL2 inhibitors (e.g., navitoclax, venetoclax), Other MDM2 inhibitors (e.g., Nutlin-3, Idasanutlin, AMG232, DS-3 032B, ALRN6924 / ATSP7041), Hypomethylating agents (HMA) (e.g., Vidaza [azacytidine, 5-azacytidine] , Dacogen [decitabine], guadecitabine), Anthracyclines (e.g., idarubicin, daunorubicin, doxorubicin, epirubicin Bishin), Anti-CD33 antibodies (e.g., Mylotarg [gemtuzumab], vadastuximab), and Beauty Other drugs (e.g., AraC [cytarabine, aracytine]) may be selected from:

[0013] The p53WT tumors which can be treated with the combination according to the invention are solid tumors or The solid tumor may be a hematological tumor. The solid tumor may be a sarcoma, such as a liposarcoma or a soft tissue sarcoma, a lymphoma, For example, non-Hodgkin's lymphoma (NHL), especially mantle cell lymphoma (MCL), melanoma, For example, cutaneous or uveal melanoma, blastoma (e.g., neuroblastoma), colon tumor, Colorectal tumors, renal tumors and liver tumors or skin cancers, such as Merkel cell carcinoma (MCC), especially The hematological neoplasm may be acute myeloid leukocyte tumor (MCC). Myeloid leukemia (AML), multiple myeloma (MM), myelodysplastic syndrome (MDS) or acute phosphorylation It may be pancreatic leukemia (ALL).

[0014] In particular, the present invention provides the following embodiments:

[0015] 1. (a) Mouse double minute 2 (MDM2) inhibitor and (b) casein kinase 1 with alpha (CK1α) degraders and / or mouse double minute 4 (MDM4) inhibitors Combinations including:

[0016] 2. (a) MD for use in treating a p53 wild-type (WT) tumor in a subject A combination comprising (b) an M2 inhibitor and a CK1α degrader and / or an MDM4 inhibitor.

[0017] 3. A method of treating a p53WT tumor in a subject, comprising administering to the subject (a) an MDM2 inhibitor administering a combination of (a) a CK1α degrader and / or an MDM4 inhibitor; The method includes:

[0018] 4. MDM2 inhibitors include Nutlin-3, Idasanutlin (RG7388, RO5503 781), RG7775(RO6839921), AMG232, DS3032(DS3 032b), ALRN-6924, ATSP-7041, CGM097 and HDM201 (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydro-pyridinyl) phenyl-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidinyl 1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]phenyl The combination of embodiment 1, embodiment 2, wherein the compound is selected from the group consisting of midazol-4-ones. The combination or method of embodiment 3 for use.

[0019] 5. MDM2 inhibitors are selected from the group consisting of Nutlin-3, Idasanutlin and HDM201. The combination of embodiment 1, the combination for use of embodiment 2 or the implementation thereof are selected from the following: Method of form 3.

[0020] 6. The combination of embodiment 1, embodiment 2, wherein the MDM2 inhibitor is HDM201. The combination for use or the method of embodiment 3.

[0021] 7. CK1α degraders and / or MDM4 inhibitors include thalidomide, pomalidomide, and lena. (RS)-3-(7-amino-3-oxo-lysine, also known as REVLIMID -1H-isoindol-2-yl)piperidine-2,6-dione and SC-24-UR 99, i.e. 4-(3-amino-1-(4-chloro-5-methyl-6-(methylamino)pyridine Lysine-3-yl)-5-fluoro-1H-indazol-6-yl)naphthalene-1- A combination of any one of embodiments 1, 4 to 6, or Combination for use of any one of embodiments 2, 4 to 6 or any one of embodiments 3 to 6 Or one way.

[0022] 8. CK1α degraders and / or MDM4 inhibitors include lenalidomide and SC-24-UR 99. Combination for use of any one of forms 2, 4 to 6 or any one of embodiments 3 to 6 One way.

[0023] 9. The MDM2 inhibitor is selected from the group consisting of Nutlin-3, Idasanutlin, and HDM201. The CK1α decomposition agent and / or MDM4 inhibitor are selected from lenalidomide and SC-24. - for the combination of embodiment 1, for the use of embodiment 2, selected from the group consisting of UR99 The combination or method of embodiment 3.

[0024] 10. The MDM2 inhibitor is HDM201, and the CK1α degrader and / or MDM The combination of embodiment 1 and the use of embodiment 2, wherein the 4 inhibitor is lenalidomide. The combination or method of embodiment 3.

[0025] 11. The method according to any one of embodiments 2, 4 to 10, wherein the p53WT tumor is a solid tumor. The combination for use in the method of any one of embodiments 3 to 10.

[0026] 12. The solid tumor is a sarcoma, such as a liposarcoma or a soft tissue sarcoma, a lymphoma, such as a non-Hodgkin's tumor. Mantle cell lymphoma (NHL), especially mantle cell lymphoma (MCL), melanoma, e.g. melanoma or uveal melanoma, blastoma (e.g., neuroblastoma), colon tumor, colorectal tumor , renal tumor, liver tumor, and skin cancer, e.g., Merkel cell carcinoma (MCC). The combination for use of embodiment 11 or the method of any one of embodiment 11.

[0027] 13. The method for use of embodiment 11, wherein the solid tumor is Merkel cell carcinoma (MCC). The combination or method of embodiment 11.

[0028] 14. Merkel cell carcinoma (MCC) is Merkel cell polyomavirus (MCV) positive The combination for use or the method of embodiment 13, wherein the combination is MCC.

[0029] 15. The p53WT tumor is a hematological tumor (or hematological malignancy), according to embodiments 2, 4-1 0 or the method of any one of embodiments 3 to 10. .

[0030] 16. Hematological tumors include acute myeloid leukemia (AML), multiple myeloma (MM), and myelodysplasia. 1. A method for treating mycobacterial leukemia comprising administering to a patient a therapeutically effective amount of a medicament for the treatment of mycobacterial leukemia or ... The combination for use of embodiment 15 or the method of embodiment 15.

[0031] 17. Hematological tumors are a group consisting of multiple myeloma (MM) and myelodysplastic syndrome (MDS). The combination for use of embodiment 15 or the method of embodiment 15, selected from:

[0032] 18. For use according to embodiment 15, the hematological tumor is myelodysplastic syndrome (MDS). The combination or method of embodiment 15.

[0033] 19. The MDM2 inhibitor is HDM201, and is a CK1α degrader and / or MDM4 inhibitor. In one embodiment, the antitumor agent is lenalidomide and the p53WT tumor is an MCV-positive MCC. The combination or method of embodiment 3 for use.

[0034] 20. The MDM2 inhibitor is HDM201, and is a CK1α degrader and / or MDM4 inhibitor. For use of embodiment 2, the antitumor agent is lenalidomide and the p53WT tumor is MDS. The combination or method of embodiment 3.

[0035] 21. The combination of any one of embodiments 1 to 20, further comprising one or more additional anticancer agents. Combinations / methods for use.

[0036] The combinations described herein provide beneficial anti-cancer effects, such as enhanced anti-cancer effects, reduced toxicity, For example, the first therapeutic agent, e.g., Any of the indicated therapeutic agents and a second therapeutic agent, e.g., one or more additional therapeutic agents, or all of the than would be required to achieve the same therapeutic effect compared to the monotherapy dose. Thus, the above-described combination therapy can be used to treat proliferation, including cancer. Kind Code: A1 Compositions and methods for treating sexual disorders are disclosed.

[0037] In some embodiments, the combinations described herein can be used to treat a subject, e.g. The methods of treating a subject with cancer described herein include administering a combination In one embodiment, the treatment regimen comprises the administration of a combination of one or more of the compounds described herein. In some embodiments, the treatment regimen includes a combination of more than one, e.g., two, three, or four. The compound has at least one and optionally two phases, e.g., a first phase and a second phase. In some embodiments, the first phase comprises a dose escalation phase. In some embodiments, the first phase may include one or more dose escalation phases, such as a first, second, or third dose escalation phase. In some embodiments, the dose escalation phase comprises, for example, In some embodiments, the combination includes administration of two, three, four or more therapeutic agents. In some embodiments, the second phase comprises a dose expansion phase. or a combination comprising two, three, four or more therapeutic agents as described herein. In some embodiments, the dose expansion phase comprises 2, 3, 4 or more doses equal to the dose escalation phase. includes more than one therapeutic agent.

[0038] In some embodiments, the first dose escalation phase comprises administering two therapeutic agents, e.g., The administration of a combination comprising two therapeutic agents, the maximum A tolerated dose (MTD) or recommended dose for expansion (RDE) is determined. Prior to the first dose escalation phase, subjects receive It was administered together with one of the therapeutic agents.

[0039] In some embodiments, the second dose escalation phase comprises administering three therapeutic agents, e.g., and administering a combination comprising three therapeutic agents, The maximum tolerated dose (MTD) or recommended dose for expansion (RDE) is determined. In some embodiments, the second dose escalation phase begins after the first dose escalation phase has ended. In some embodiments, the second dose escalation phase may include administering one or more of the therapeutic agents administered in the first dose escalation phase. In some embodiments, the second dose escalation phase comprises administering the above doses after the first dose escalation phase. It is carried out without any enforcement.

[0040] In some embodiments, the third dose escalation phase comprises four therapeutic agents, e.g., The present invention also includes administering a combination comprising four therapeutic agents, wherein one, two, three or all of the therapeutic agents are administered simultaneously. The largely tolerated dose (MTD) or recommended dose for expansion (RDE) is determined. In some cases, the third dose escalation phase begins after the first or second dose escalation phase is completed. In an embodiment, the third dose escalation phase comprises administering a dose of the therapeutic agent administered in the second dose escalation phase. In some embodiments, the third dose escalation phase comprises administering one or more (e.g., all) of In some embodiments, the first dose escalation phase includes administration of one or more of the therapeutic agents administered in the first dose escalation phase. In the present study, the third dose escalation phase was conducted without conducting the first, second, or both dose escalation phases. It will be carried out.

[0041] In some embodiments, the dose expansion phase begins after the first, second, or third dose escalation phase has ended. In some embodiments, the dose expansion phase begins after a dose escalation phase, e.g., a first, second or third dose. comprises administering the combination in a third dose escalation phase. Biopsies are obtained from subjects in the dose expansion phase.

[0042] Without wishing to be bound by theory, in some embodiments, the dose escalation phase and dose expansion Treatment regimens that include phases include the introduction of new agents or regimens for combination, combination Allows for rapid generation of combinations and / or evaluation of safety and activity of acceptable combinations. It is thought that this is the case.

[0043] Here, we demonstrate that MCV ST functions as a transcriptional activator and then mediates M Increased levels of MDM2 and CK1α, which cooperate with DM4, inhibited p53 function in MCC. The present inventors demonstrate that MDM2 and MDM4 are involved in the expression of both MDM2 and MDM4 in MCC. This further demonstrates the synergistic efficacy of targeting both

[0044] Merkel cell carcinoma (MCC) is an aggressive type of skin cancer. Myomavirus (MCV) is a mycoplasmic cancer cell that contains inactivating mutations in RB and p53. On the other hand, MCV-positive MCCs usually contain wild-type RB and p53. Binding of MCV large T antigen to M leads to p53 activation, whereas binding of MCV small T antigen to M By increasing the levels of DM2 and CK1α, an activator of MDM4, p53 We demonstrate that lenalidomide or a specific MDM4 inhibitor reduces CK activation. Targeted degradation of 1α synergizes with MDM2 inhibitors to activate p53 and inhibit apoptosis. Our study elucidates the mechanism behind MCV regulation of p53 in MCC. and combinatorial targeting of MDM2 and MDM4 in p53 wild-type tumors. Demonstrate the usefulness of this.

[0045] Merkel cell polyomavirus (MCV) is responsible for all Merkel cell carcinomas (MCC), MCV-positive MCC accounts for approximately 80% of highly aggressive neuroendocrine carcinomas of the skin. They express truncated forms of the T antigen (ST) and large T antigen (LT), and usually express wild-type p5 3 (TP53) and RB (RB1). In contrast, virus-negative MCCs contain TP 53 and contains an inactivating mutation in RB1. MCV truncated LT binds to RB. The present inventors have now discovered that MCV L can bind to and inhibit p53, but does not bind to p53. T binds to RB, leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, co-expression of ST reduced p53 activation.

[0046] MCV ST mediates the transcription of the MYC homolog MYCL (L-Myc) by EP400 chromatin ribosomal transcription. The inventors have demonstrated that MCs recruit β-terminally activated ... Depletion of EP400 in V-positive MCC cell lines leads to increased p53 target gene expression We observed that the MCV ST-MYCL-EP400 complex inhibits p53 function. Although it was suspected that EP400 could be selectively inactivated, the underlying mechanism was unknown. Integrated ChIP and RNA-seq analysis after depletion demonstrated that MDM2, CK1α, and MDM4 are activated We identified activator as a target gene of the ST-MYCL-EP400 complex. CV-positive MCC cells expressed high levels of MDM4. Combining with lenalidomide or MDM4 inhibitors targeting p53 synergistic activity in MCV-positive MCC cells and in MCC-derived xenografts in mice. These results suggest that viral-positive MCC and other p53-related Supports dual targeting of MDM2 and MDM4 in live tumors. [Brief description of the drawings]

[0047] [Figure 1-1]Figure 1: Merkel cell polyomavirus large T antigen activates and small T antigen attenuates the p53 response. A. Inducible expression of truncated tumor isoforms of MCV LT increases ARF and p53 target genes in IMR90 cells. Expression of GFP or LT-L21 and LT-162 truncated LT was induced by doxycycline (DOX) treatment for 24 h. LT, ARF and p53 target gene RNA levels were normalized to those of GFP-induced cells, while GFP levels were normalized to LT-L21 samples. Data are shown as mean ± SD; *Student's t test P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005. B. IMR90 cells were induced to express GFP, ST, LT-L21 or LT-L21 together with ST for 40 h. Lysates were prepared before (-) or after (+) DOX. Activation of the p53 response is reflected by increased levels of p53, phospho-serine 15 p53 (P-p53), acetyl-lysine 382 p53 (Ac-p53), p21, and cleaved PARP (**). C. Expression of L21 activates p53 through inhibition of RB and induction of ARF, whereas a LT mutant (E216K) in the LXCXE motif does not activate it. [Figure 1-2] (As stated above.) [Figure 1-3] (As stated above.) [Figure 2-1]Figure 2: MDM2 and CK1α are transcriptional targets of the ST-MYCL-EP400 complex. A. Volcano plot illustrating differentially expressed p53 target genes in MKL-1 MCC cell line after depletion of EP400 with inducible shRNA versus control shRNA. Each gene log2 fold change was plotted against -log10 p-value for statistical significance. Green dots indicate genes meeting a 2-fold change cutoff, red dots mean adjusted p-values ​​less than 0.1. B. RT-qPCR was performed after 8 days of shRNA induction on MKL-1 cells. Reads were normalized to RPLP0 and uninduced samples. Experiments were performed in triplicate and averaged. Data are shown as mean ± SD; *Student t-test P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005. C. ChIP with MAX, EP400, ST and IgG antibodies of the indicated promoters in MKL-1 cells followed by pPCR. ChIP-qPCR was performed in triplicate and the average percent input is shown. D. Depletion of MCV T antigen causes a reduction in MDM2, CK1α and MDM4 levels. MKL-1 cells were transduced with specific shRNA for 5 days and harvested for Western blotting. E. ChIP-qPCR with MAX, EP400 and ST antibodies for the MDM2 promoter in IMR90 cells in the presence (+) or absence (-) of MCV T antigen. ChIP was performed five times independently. F. Nutlin-3 treatment does not induce a p53 response in IMR90 cells expressing p53DD, whereas MCV T antigen increases the levels of MDM2, MDM4 and CK1α. MKL-1 and IMR90-p53DD were treated with Nutlin-3 (1 μM) for 24 h. [Figure 2-2] (As stated above.) [Figure 2-3] (As stated above.) [Figure 2-4] (As stated above.) [Figure 2-5] (As stated above.) [Figure 2-6] (As stated above.) [Figure 3-1]Figure 3: MDM4 is overexpressed in MCV-positive MCC. A. RNA from MCC cell lines and human foreskin fibroblasts (HFFs) was harvested for RT-qPCR for MDM4 (total), MDM4-FL (full-length variant) and MDM4-S (short splice variant). MDM4 levels were normalized to the geometric mean of RPLP0, 18s rRNA and beta-actin RNA controls. Data shown as mean ± SD; *Student's t-test P<0.05 for MDM4-FL. B. Western blot of MCC cell lines and HFFs with the indicated antibodies. MS-1 and MCC13 overexpress p53 due to an inactivating mutation, while MKL-2 and MCC26 do not express detectable levels of p53. [Figure 3-2] (As stated above.) [Figure 4-1]Figure 4: Inhibition of MDM2 and MDM4 enhances p53 activation in MCC cell lines. A. Lenalidomide enhances p53 activation by Nutlin-3 in MKL-1. MKL-1 cells were treated with Nutlin-3 (5 μM), Lenalidomide (Len, 10 μM) or both for 40 hours. B. Lenalidomide depletes CK1α but does not enhance p53 activation by Nutlin-3 in UISO cells. MKL-1 (MCV+MCC) or UISO (MCV-MCC) cells were treated with Nutlin-3 (1 μM) with or without Lenalidomide (10 μM) for 24 hours. Notably, UISO has less MDM2 and MDM4 protein than MKL-1. C. Lenalidomide and to a lesser extent pomalidomide, but not thalidomide, cooperate with Nutlin-3 to activate p53. MKL-1 cells were treated with Nutlin-3, lenalidomide, pomalidomide (10 μM) or thalidomide (10 μM) for 24 hours. D. Lenalidomide treatment reduces MDM4 binding to p53 and activated MDM2. MKL-1 cells were treated with Nutlin-3 (5 μM), lenalidomide (10 μM) or both for 40 hours and harvested for immunoprecipitation with antibodies against MDM4, p53 and CK1α followed by Western blotting. E. Depletion of CK1α by CRISPR transduction enhances p53 activation by Nutlin-3. MKL-1 cells stably expressing each of the two CK1α sgRNAs were treated with Nutlin-3 (1 μM) with or without lenalidomide for 24 hours. Lenalidomide further reduced CK1α that the sgRNAs did not completely deplete. F. MDM4 inhibitor SC-24-UR99 (UR99) cooperates with MDM2 inhibitors in activating p53. MKL-1 cells were treated with Nutlin-3 (1 μM) or HDM201 (0.1 μM) with or without lenalidomide (1 μM) or UR99 (0.1 μM). [Figure 4-2] (As stated above.) [Figure 4-3] (As stated above.) [Figure 4-4] (As stated above.) [Figure 4-5] (As stated above.) [Figure 4-6] (As stated above.) [Figure 5-1] Figure 5: Inhibition of MDM2 and CK1α-MDM4 synergistically induces apoptotic cell death. A. MKL-1 and MS-1 cells were treated with MDM2 inhibitors Nutlin-3, RG7388 or AMG232 at several concentrations and XTT assays were performed 96 hours after treatment. **Multiple t-test p-value<0.005. B. Bliss synergy studies show strong synergy between Nutlin-3 and Lenalidomide or SC-24-UR99, but not Thalidomide. C. BH3 profiling was performed with MKL-1 cells treated with Lenalidomide, Nutlin-3 or both drugs for 16 hours. Experiments were performed in triplicate. Data are shown as mean ± SD; *Student t-test P<0.05. D. MKL-1 MCC xenografts in SCID mice responded to combination treatment with HDM201 and lenalidomide. HDM201 (40 mg / kg), lenalidomide (50 mg / kg) or both drugs were orally administered daily starting when xenograft tumors were 200 mm3. Data are shown as mean ± SEM; multiple t-test between HDM201 and combination treatment *P<0.05# - Study was terminated because tumor volume reached maximum allowable size. E. Model: Merkel cell polyomavirus T antigen sensitizes Merkel cell carcinoma for targeting of the p53-MDM2-MDM4 pathway. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 5-4] (As stated above.) [Figure 5-5] (As stated above.) [Figure 6-1]Figure 6: (also referred to herein as Figure S1). A. Inducible expression of full-length or truncated tumor isoforms of MCV LT activates p53 in IMR90 cells. Expression of GFP or WT (wild type) LT, FL (full-length) LT, 162, 168 and L21 truncated LT was induced by doxycycline (DOX) treatment for 40 hours. Lysates were prepared before (-) or after (+) DOX. Activation of the p53 response is reflected by increased levels of p53, phospho-serine 15 p53 (P-p53), acetyl-lysine 382 p53 (Ac-p53), p21 and cleaved PARP (**). B. RT-qPCR shows that LT-L21 induction in IMR90 cells increases the levels of the known p53 target gene p21. L21-LT was induced for 4, 8, 12, 16, 20, 24, 36, 48, 60, and 72 hours with ST alone or as a splice variant, and RNA was harvested for RT-qPCR. Data shown are representative of three independent experiments. **Two-way ANOVA p-value <0.005. C.LXCXE LT mutants can bind VPS39 but not RB. HCT116 cells stably expressing GFP, LT-L21, or LT-L21 E216K mutant were harvested for LT immunoprecipitation. [Figure 6-2] (As stated above.) [Figure 6-3] (As stated above.) [Figure 7-1]Figure 7: (also referred to herein as Figure S2). A. RNA-seq normalized counts show that EP400 shRNA reduced levels of MDM2 and increased p21 compared to control shRNA in MKL-1. B. RNA-seq fold change of MDM2, CK1α (CSNK1A1) and EP400 compared to control shRNA after depletion of EP400 using two independent shRNAs shows reduced levels of MDM2 and CK1α along with EP400. Adjusted p-values ​​for Bonferroni correction are shown. C. MKL-1 cells were induced to express EP400 or control shRNA for 8 days. Western blot shows reduced MDM2 and EP400 levels and increased p53 and p21 levels with EP400 shRNA. D. IMR90 cells stably expressing hTERT, p53DD, MYCL (IMR90-p53DD) with (+) or without (-) MCV T antigen (LT-L21 and ST) were blotted with the indicated antibodies. E. Two independent ChIP-seq of MAX, EP400, and ST show peaks in the MDM2 and CK1α promoters, also marked by H3K4me3. F. ChIP-qPCR of ST shows that MCV ST binds to the MDM2 and CK1α promoters in IMR90-p53DD cells expressing MCV T antigen. Experiments were performed in duplicate. Data are shown as mean ± SD; *P<0.05, ns>=0.05. G. ST and EP400 enrichment in the MDM2 and CK1α promoters in MKL-1 cells depleted of p53. MKL-1 stably expressing p53 shRNA was used. Experiments were performed in duplicate. [Figure 7-2] (As stated above.) [Figure 7-3] (As stated above.) [Figure 7-4] (As stated above.) [Figure 7-5] (As stated above.) [Figure 7-6] (As stated above.) [Figure 7-7] (As stated above.) [Figure 8-1]Figure 8: (also referred to herein as Figure S3). A. Ratio of MDM4-FL (full length) to MDM4-S (short version). B. cBioPortal analysis of 101 cases of MCC reveals 37 tumors with TP53 and RB1 mutations and high levels of mutations compared to 64 MCCs with no TP53 or RB1 mutations. MDM2 and MDM4 show frequent copy number gain (3-8 copies) and amplification (>8 copies). [Figure 8-2] (As stated above.) [Figure 9-1] Figure 9: (also referred to herein as Figure S4). A. Lenalidomide rapidly reduces CK1α protein in MKL-1. MKL-1 cells were treated with lenalidomide (Len, 10 μM) and / or cycloheximide (CX, 5 μM) for 2, 4 or 6 hours with or without MG132 (10 μM). p53 is a positive control for cycloheximide pulse-chase treatment. B. MKL-1, WaGa, Peta, BroLi and MS-1 MCV-positive MCC cell lines were treated with nutlin-3 (5 μM) with or without lenalidomide (10 μM) for 40 hours. C. Lenalidomide (10 μM) and to a lesser extent pomalidomide (10 μM), but not thalidomide (10 μM), reduce CK1α protein levels. D. MKL-1 cells were treated with Nutlin-3, Lenalidomide or both for 16 hours followed by cycloheximide treatment for 2 and 4 hours. Western blot bands were quantified using LiCor and normalized to the 0 hour time point for each treatment. p53 half-life is estimated to be 1.5 hours for DMSO and Lenalidomide treated samples and 4 hours for Nutlin-3 treated samples. Experiments were performed in triplicate and data are shown as mean ± SD; ** Two-way ANOVA < 0.005. E. MKL-1 cells were treated with Nutlin-3 (5 μM), Lenalidomide (10 μM) or both for 24 or 40 hours and harvested for IP-Western blotting with MDM4, p53 and CK1α. [Figure 9-2] (As stated above.) [Figure 9-3] (As stated above.) [Figure 9-4] (As stated above.) [Figure 9-5] (As stated above.) [Figure 10-1] Figure 10: (also referred to herein as Figure S5). A-C. MKL-1 cells were treated with Nutlin-3 (A), RG7388 (B) or AMG232 (C) with or without lenalidomide (fixed concentration of 5 μM). XTT was performed at 96 hours of treatment. D. MKL-1 (with wild type p53) and MS-1 (with p53 mutation) were treated with Nutlin-3 with or without lenalidomide for 96 hours and XTT assays were performed to measure relative cell viability normalized to DMSO control. E. Lenalidomide and to a lesser extent pomalidomide, but not thalidomide, synergize with Nutlin-3 in reducing MKL-1 viability. XTT assays were performed with three biological replicates at 96 hours of treatment. Data are shown as mean ± SD; *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 and ns>=0.05. [Figure 10-2] (As stated above.) [Figure 10-3] (As stated above.) [Figure 10-4] (As stated above.) [Figure 10-5] (As stated above.) [Figure 11-1] Figure 11: (also referred to herein as Figure S6). A. Compusyn synergy study shows that Nutlin-3 and RG7388 synergize with lenalidomide. XTT assay was performed after 96 hours of treatment of MKL-1 cells. A CI value less than 1 indicates synergy. B-D. Dose response matrix of Nutlin-3 with thalidomide (B), lenalidomide (C) or SC-24-UR99 (D). E. ZIP synergy study. F. HSA synergy study. [Figure 11-2] (As stated above.) [Figure 11-3] (As stated above.) [Figure 11-4] (As stated above.) [Figure 11-5](As stated above.) [Figure 11-6] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] LT activates and ST attenuates the p53 response. To test the effect of MCV T antigen on p53 in normal cells, GFP or LT were used. Doxycycline-inducible vectors expressing tumor-derived truncated or full-length forms were The truncated forms of LT were introduced into MR90 diploid lung fibroblasts (Fig. 1A, S1A). As examples, L21 (residues 1 to 292) contains an intact LXCXE motif. code), 162 (residues 1-320), and 168 (residues 1-275).[6] LT-L21 and LT-162 expression in IMR90 was assessed by RT-qPCR. , ARF, and several p53 target genes, including GDF15 and p21 (CDKN1A). Inhibition of RB significantly increased the levels of E2F transcription factors (Fig. 1A). This leads to increased levels of ARF, the major p53-degrading E3 ligase.

[10] It is a potent inhibitor of MDM2.

[14] LT expression is indicative of p53 and p53 activation. Increased the protein levels of phospho-serine 15p53 (P-p53) and p21 ( S1A). The expression of LT-162 also increased the cleaved PARP ( ** ) levels, resulting in an increase in Apoptosis The results showed a systolic response.

[0049] LT and ST are the serovars that are secreted from integrated MCPyV viral DNA in MCC tumors. To mimic this in the IMR90 cell line, To this end, we introduced a genomic version of LT-L21 that co-expresses ST. When T was co-expressed with LT-L21, the response was lower compared with that to LT-L21 alone Levels of p53 activation (p21, p-p53 and acetyl-lysine 382 or AC-p5 3) was observed (Fig. 1B, S1B). This result indicates that truncated LT activates p53. We show that ARF can enhance the expression of IFN-γ in the IL-1 / IL-2 ... To determine whether the increased levels of L required LT binding to RB, we A point substitution mutation (E216K) was introduced in the LXCXE motif of T-L21. When stably expressed in 116 cells, L21-E216K was unable to co-precipitate RB. However, it retained binding to VPS39, which binds to a different region of LT (S1C) [5]. When expressed in IMR90 cells, L21-E216K reduced the levels of ARF and p21 It did not increase the levels of p53, AC-p53, or IL-1 (Fig. 1C ). These results suggest that LT binding to RB leads to increased levels of ARF and activation of the p53 response. Notably, when ST was co-expressed with L21-E216K, , increased levels of MDM2, but not p21, were detected.

[0050] MDM2 and CK1α are transcriptional targets of the ST-MYCL-EP400 complex. The present inventors have demonstrated that MCV ST binds the MYC homolog MYCL (L-Myc) to the EP400 cluster. They then recruit the chromosome remodeler complex to bind to specific gene promoters and regulate their expression. Recently, we reported that ST-MYCL-E activates the MYCL-1 gene in MKL-1 cells. Three different shRNAs were used to identify genes regulated by the P400 complex. We performed RNA-seq after depletion of EP400, which is a marker for mitochondrial endothelial cell death

[15] . Using the results, we assessed changes in gene expression of known p53 target genes. [9]. EP400 depletion significantly reduced the expression of p21(CD11) as visualized by volcano plots. This resulted in increased levels of many p53 target genes, including KN1A (Figure 2A). Using a fold change cutoff of 5, 59 of a total of 198 p53 target genes were identified. 17 genes were upregulated and 17 were downregulated (Table S1) [9]. This resulted in a decrease in the levels of MDM2E3 ligase and CK1α (Figures 2B, S2A-C MDM2 and MDM4 directly bind to the transactivation domain of p53 and inhibit p53 activity. MDM4 contains an N-terminal p53-binding domain that blocks activation of p53

[11] . The N-terminal p53-binding domain of MDM4 is involved in the regulation of its own activity. CK1α (CSNK1A) binds to MDM4, phosphorylates it, and then binds to p53, forming an intramolecular interaction with the W motif region, thereby reducing its binding to p53

[0012] . It is a serine / threonine kinase that prevents the autoinhibitory interaction of β-lactamase with β-lactamase and activates MDM4.

[13] RT-qPCR and Western blotting showed that EP Upon 400 knockdown, p21 levels increased and MDM2 and CK1α levels decreased This was confirmed (Figs. 2B and S1C).

[0051] ChI using antibodies against ST, MAX (which dimerizes with MYCL), and EP400 P-seq revealed enrichment of the MDM2 and CK1α promoters (S2E)

[0015] . We performed ChIP-qPCR for MAX, ST, and EP400. We observed specific enrichment of these promoters (Fig. 2C). We depleted ST or ST and LT in cells using specific shRNAs, and We found that the level of K1α reduced the expression of shRNA (Figure 2D)

[16] . The results, together with the RT-qPCR and ChIP data, indicate that MDM2 and CK1α are ST- These results suggest that MYCL-EP400 is a direct transcriptional target of the MYCL-EP400 complex. Although MDM4 levels were reduced upon ST depletion, we hypothesized that the direct upregulation of MDM4 by ST We found no evidence for specific activation of MDM4, which contributes to p53. It increases the levels of CK1α which may function to activate its activity.

[0052] Since MDM2 is a p53 target gene, MCV T antigen is induced by p53 activation. It is possible that MDM2 levels may be increased indirectly [9]. To exclude this possibility, Therefore, the present inventors have isolated a p53 dominant gene that binds to and inactivates endogenous p53. The active version (p53DD) was introduced into IMR90 cells

[17] . -p53DD cells were further transduced with MCV LT-L21 together with MYCL and ST.

[17] . We detected ST binding to the MDM2 and CK1α promoters by ChIP. - EP400 enrichment for the MDM2 promoter was detected by qPCR using MCV T antibody We observed that MDM2 expression was increased in the presence of nutlin-3 (Fig. 2E, S2F). Inhibitor treatment did not increase p53, p-p53, and PUMA levels, indicative of p53 activation. The ST-MYCL-EP400 complex was found to be p53-independent. To further determine whether s We depleted p53 using hRNA and performed ChIP-qPCR for EP400 and ST. (S2G). We found that p53 exerts an EP These results suggest that MCV ST does not affect MC 400 or ST concentrations. Transactivates MDM2 and CK1α in a p53-independent manner in C and IMR90 cells. This indicates that.

[0053] MDM4 is overexpressed in virus-positive MCC. Overexpression of MDM4 can be found in some cancers with wild-type p53. We detected MDM4 in MCC cell lines using three sets of MDM4 primers. Total MDM4 (all splice variants), MDM4-FL (full length) and MDM4-S (short variant lacking the RING domain) levels were assessed

[18] . (MKL-1, MKL-2, MS-1, WaGa, PeTa and BroLi) MCC cells The cell lines were significantly more aggressive than the virus-negative (UISO, MCC13, and MCC26) MCC lines. They had high levels of total MDM4 and MDM4FL (Figure 3A). The ratio of MDM4-S was higher in the virus-positive line (S3A). MDM4-FL protein levels were assessed in these MCC cell lines. C cell lines expressed higher levels of MDM4-FL compared with virus-negative MCC cell lines. Notably, the virus-negative UISO cell line expressed high levels of MDM4-FL. and MDM4-S, but did not express abundant levels of MDM4 protein. (Fig. 3A, S3A). LT activation of p53 in virus-positive MCC was associated with MDM2 or Can create a dependency on MDM4 expression. Targeted next-generation sequencing of 101 MCC tumors The Oncopanel CT scan showed that despite the presence of MCV, -gain (3 to 8 copies) (S3B)

[19] .

[0054] Inhibition of MDM2 and MDM4 activates p53 in MCC. The present inventors have demonstrated that MKL-1, WaGa, PeTa and BroL contain wild-type p53. i The levels of total p53, P-p53, Ac-p53, p21 and PUMA in MCC cell lines Nutlin-3 MDM2 inhibitor treatment activates p53 as indicated by increased but not in MS-1 cells harboring an inactivating p53 mutation. (Fig. 2F, Fig. 4A, S2B)

[20] . The ligase could specifically target CK1α for ubiquitination in the presence of lenalidomide We demonstrated that lenalidomide inhibits CK1 in MCC cells. We assessed whether MKL-1 cells could be transformed with Leucine-α to reduce p53 levels and activate p53. Blocking protein synthesis with lidomide, treated with or without cycloheximide CK1α levels were not significantly altered by 6-hour cycloheximide treatment. However, levels rapidly decreased after the addition of lenalidomide (S4A).

[0055] Lenalidomide treatment alone had a modest positive effect on p53 levels. However, when Nutlin-3 and lenalidomide were combined, p53 and p53 target genes A greater increase in p53 expression was observed in cell lines with wild-type p53 (Figures 4A, S4B ) We have demonstrated by quantitative Western blotting that in the presence of cycloheximide p5 in MKL-1 cells treated with nutlin-3, lenalidomide, or both 3Stability was evaluated (S4D). Lenalidomide inhibited p53 stability in the presence of Nutlin-3. 23 Depletion of CK1α by lenalidomide significantly increased the activity of p53. The present inventors have demonstrated that lenalidomide reduces the activity of MDM2 and MDM4, which are involved in the activation of MDM1 and MDM2. p53 activation in virus-negative and p53 wild-type UISO cells that barely express p53 4 We examined whether lenalidomide enhances UISO cell proliferation (FIGS. 3A-B). Reduces CK1α levels in cells but fails to enhance p53 activation by Nutlin-3 We observed that the α-amyloid β ...

[0056] Structural analysis of CRBN revealed that lenalidomide can potently promote the interaction with CK1α However, the related compounds thalidomide and pomalidomide are less able to recruit CK1α to CRBN.

[22] Compared with lenalidomide, pomalidomide and thalidomide The aldehyde did not reduce CK1α protein levels in MKL-1 cells (S4C). In addition, pomalidomide or thalidomide inhibited p53 expression against nutlin-3 in MKL-1 cells. The contribution of CK1α to MDM4 binding to p53 was not enhanced by CK1α alone (Fig. 4C). To test this, we treated mice with Nutlin-3 and lenalidomide for 40 hours. Lysates prepared from MKL-1 cells were immunofluorescentlyzed for MDM4, p53, and CK1α. Immunoprecipitation (IP) was performed (Figs. 4D and S4E). Lenalidomide and Nutlin-3 inhibited the nuclease activity of nuclease-binding proteins. It reduced CK1α levels and increased p53 levels compared to Trin-3 alone. Despite increased levels of nutlin-3, lenalidomide co-treatment with nutlin-3 was significantly higher than untreated or nutlin-3. Co-regulation of p53 by MDM4, CK1α or activated MDM2 compared with Torin-3-treated controls The reduction in CK1α levels did not increase cytoplasmic levels of IL-1 or activate MDM2. 53.

[0057] If CK1α enables MDM4 binding to p53, we hypothesize that loss of CK1α We predicted that MDM4 binding to p53 would be reduced and p53 activation enhanced

[0013] . To test this, we performed a multicenter study using two independent CRISPR sgRNAs. We depleted CK1α in MKL-1 and assessed p53 activation after Nutlin-3 treatment ( Figure 4E)

[24] . CRISPR knockout of CK1α reduces the expression of activated MDM2 (phospho- Serine 166, P-MDM2), P-p53, p53 and p21 levels. The results showed that CK2 expression was significantly increased by either lenalidomide or sgRNA, resulting in increased p53 activity. Reduction of 1α has been shown to enhance p53 activation

[25] . In particular, lenalidomide Addition of CK1α reduced protein levels that were not completely depleted by CRISPR. The inventors used a newly developed MDM4 inhibitor to further increase the p53 activation marker. The inhibitors (SC-24-UR99, UR99) were either MDM2 inhibitor Nutlin-3 or HDM20 We then tested whether UR99 could enhance the activity of p53-associated IL-1 (Figure 4F)

[26] . Specifically blocks MDM4 binding. We show that inhibition of MDM4 by UR99 inhibits MDM4 binding. We found that it enhanced p53 activation by M2 inhibitors.

[0058] Lenalidomide synergizes with MDM2 inhibitors to induce apoptosis in MCC cell lines do. To demonstrate the effect of MDM2 inhibition on MCC cell viability, we We treated MS-1 (p53 wild-type) and MS-1 (p53 mutant) cells with the MDM2 inhibitor Nutlin 3, RG7388 or AMG232 for 96 hours and then subjected to XTT viability assay. MKL-1 was sensitive to all three MDM2 inhibitors, but not to MS- 1 cells were not sensitive (Figure 5A). To test the effect of combining RG7388 and AMG232 with lenalidomide We observed significantly improved cytotoxicity of all three MDM2 inhibitors when combined with ~D). Synergism using Compusyn (S6A) and Bliss (Figure 5B) methods The study examined the combination of Nutlin-3 or RG7388 with lenalidomide or UR99. In contrast, thalidinol showed synergistic activity against thalidinol (S6B-F) [28-31]. MIDE has evidence of synergy when used in combination with Nutlin-3. CK1α levels, consistent with its relatively reduced effect on CK1α levels (Figure 5B, S 5E).

[0059] Lenalidomide induces apoptotic cell death in MCC cells using Nutlin-3 To determine whether this affected the ability of the mice to express BH3 peptides, we We performed BH3 profiling to measure the sensitivity of lenalid to Nutlin-3

[32] . The addition of amide enhanced the priming effect for apoptosis (Figure 5C). To determine the dual inhibition of MDM2 and MDM4 in vivo, we used MKL-1 M CC xenografts were treated with HDM201 (suitable for in vivo efficacy testing) to induce lenalidomide The present inventors found that the addition of lenalidomide significantly increased the IL-1 expression in the IL-1–induced ... Significantly enhances the efficacy of HDM201 in p53 wild-type mice expressing MDM2 and MDM4 The findings provide potential clinical benefit for combination therapy for tumors. We found that MCV T antigen suppresses p53 activity in virus-positive MCC. Model showing therapeutic potential by increasing dependency on MDM2, MDM4 and CK1α for We propose (Figure 5E).

[0060] Lenalidomide is used to treat malignant tumors, myelodysplastic syndromes (MDS) and multiple myeloma (MM). Mutant CSNK1A1 has been used to treat lenalidomide. It has been reported that mutations in the IL-1 gene predict poor prognosis in patients with MDS

[33] . MDS patients with p53 responded better to lenalidomide than those with wild-type p53 and there is a high possibility of progression to acute myeloid leukemia. Our study shows that this may be partially dependent on inactivation of MDM4

[34] . in MCC containing wild-type p53 and in other solid tumors and hematological malignancies. This study provides a rationale for combining lenalidomide with an MDM2 inhibitor.

[0061] For human use, lenalidomide is available, for example, in capsule form as REVLIMID. For example, for MM combination therapy, days 1-21 of repeated 28-day cycles 25 mg orally once daily; 1 of repeated 28-day cycles for MM maintenance therapy after autologous HSCT Continue at 10 mg once daily for up to 28 days; 10 mg once daily for MDS; May be administered orally at 25 mg once daily on days 1-21 of repeated 28-day cycles. Medication should be continued or modified based on clinical and laboratory findings in cases of renal injury. Doses are adjusted based on creatinine clearance values.

[0062] For human use, HDM201 can be administered orally, for example, in capsule form. In particular, oral administration is recommended for high-dose intermittent regimens [e.g., Regimen A (50 mg to 400 mg)]. mg of HDM201 administered on day 1 of a 3-week cycle or Regimen B (50 mg to 150 mg) mg of HDM201 administered on days 1 and 8 of a 4-week cycle) or Regimen C (50 mg ~500 mg HDM201 on day 1 of a 4-week cycle] or a low-dose extended regimen [For example, Regimen D (10 mg to 30 mg of HDM201 in the first 2 weeks of a 4-week cycle) 1 week, once daily) or Regimen E (15 mg to 50 mg of HDM201 in a 4-week cycle) (once per day for the first week of treatment) may be used.

[0063] For human use, idasanutlin may be administered, for example, in capsule form for 28-day treatment cycles. It can be administered orally once or twice daily on days 1 to 5 of the first week of the month. The dosage may be from 00mg to 1000mg.

[0064] For human use, SC-24-UR99 may be administered intravenously, for example in liquid or capsule form. Administration can be by intravenous or oral administration. 10mg to 1000mg of SC-24-UR99 was administered on day 1 of a 3-week cycle or Zymen B (5 mg to 500 mg of SC-24-UR99 on days 1 and 8 of a 4-week cycle Regimen C (100 mg to 1000 mg of SC-24-UR99 administered for 4 weeks) on day 1 of ipilimumab] or low-dose extended regimen [e.g., regimen D (1 mg to 100 mg mg SC-24-UR99 once daily for the first 2 weeks of a 4-week cycle) or E(1 mg to 200 mg of SC-24-UR99, once daily during the first week of a 4-week cycle) )] may be used.

[0065] chemical formula The compounds listed herein may also be represented by the following chemical formula: [ka]

[0066] table

[0067] [Table 1]

[0068] [Table 2]

[0069] [Table 3]

[0070] [Table 4]

[0071] definition As used herein, the articles "a" and "an" refer to the Refers to one or more (e.g., at least one) of the grammatical referents of a word.

[0072] The term "or" is used herein to mean "and / or" unless the context clearly dictates otherwise. "or" is used interchangeably herein.

[0073] "About" and "approximately" generally refer to the quantity measured given the nature or precision of the measurements. An exemplary degree of error is given by: Within 20 percent (%) of a value or range of values, typically within 10% and more typically It is within 5%.

[0074] As used herein, the articles "a" and "an" refer to the Refers to one or more (e.g., at least one) of the grammatical referents of a word.

[0075] The term "or" is used herein to mean "and / or" unless the context clearly dictates otherwise. "or" is used interchangeably herein.

[0076] "About" and "approximately" generally refer to the quantity measured given the nature or precision of the measurements. An exemplary degree of error is given by: Within 20 percent (%) of a value or range of values, typically within 10% and more typically It is within 5%.

[0077] "Combination" or "in combination with" means that the therapies or treatments are administered at the same time. and / or must be formulated for delivery together. Although it is not intended to imply this, such delivery methods are described herein. The therapeutic agent in the combination may be one or more other additional therapies or treatments. The therapeutic agent or protocol may be administered simultaneously with, prior to, or subsequent to the administration of the therapeutic agent. They may be administered in any order. Generally, each agent will be administered according to the schedule determined for that agent. In addition, the combination will be administered at a dose and / or time schedule that is beneficial for The additional therapeutic agents used may be administered together in a single composition or separately in different compositions. It will be understood that additional therapeutic agents utilized in combination generally may be administered. It is expected that each of these will be used at a level not in excess of the level at which it is used individually. In some embodiments, the levels utilized in combination are higher than the levels utilized individually. will also be lower.

[0078] In embodiments, the additional therapeutic agent is administered in a therapeutic or sub-therapeutic amount. In some embodiments, the concentration of the second therapeutic agent required to achieve inhibition, e.g., growth inhibition, is In certain embodiments, the IL-1 receptor agonist activity is lower when the second therapeutic agent is administered in combination with the first therapeutic agent. In the present study, the concentration of the first therapeutic agent required to achieve inhibition is When administered in combination with a first therapeutic agent, the level of In certain embodiments, the combination therapy requires a second therapeutic agent to achieve inhibition. The concentration of the second therapeutic agent is lower than the therapeutic dose of the second therapeutic agent as a monotherapy, for example, 10-20%, 2 0~30%, 30~40%, 40~50%, 50~60%, 60~70%, 70~80% or 80-90% lower. In certain embodiments, the combination therapy achieves inhibition The concentration of the first therapeutic agent required for this is lower than the therapeutic amount of the first therapeutic agent as a monotherapy. , for example 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 6 0-70%, 70-80% or 80-90% lower.

[0079] The terms "inhibition," "inhibitor," or "antagonist" refer to an agent that inhibits the activity of a given molecule, e.g., an immune checkpoint. The specific parameters of the cross-linker inhibitors, such as a reduction in activity, include, for example, at least 5 %, 10%, 20%, 30%, 40% or more of the activity, e.g., of a given molecule. Inhibition, e.g., inhibitory molecules, are included in this term. Thus, inhibition does not necessarily have to be 100%. stomach.

[0080] The term "anti-cancer effect" refers to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of tumor metastases, etc. reduction in cancer cell proliferation, reduction in cancer cell survival or various other conditions associated with the cancerous condition The effects of the treatment may be manifested by a variety of means, including, but not limited to, amelioration of certain physiological symptoms. "Anti-cancer effect" refers to the biological effect of preventing the onset of cancer in the first place. It may also be demonstrated by the capacity of peptides, polynucleotides, cells and antibodies.

[0081] The term "antitumor effect" refers to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, This can be demonstrated by a variety of means, including, but not limited to, a reduction in tumor cell proliferation or a reduction in tumor cell survival. The term refers to a biological effect that can be demonstrated.

[0082] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body, either locally or through the bloodstream and lymphatic system. Examples of various cancers are described herein, including breast cancer, prostate cancer, ovarian cancer, cervical cancer, These include skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. The terms "tumor" and "cancer" are used interchangeably herein. For example, both terms encompass solid and liquid, e.g., disseminated or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-cancerous as well as malignant cancers. As used herein, the term "cancer" refers to the formation of a primary malignant cell or tumor ( For example, the cells have not metastasized to sites within the subject's body other than the site of the original malignant tumor or tumor. and secondary malignant cells or tumors (e.g., secondary sites different from the site of the original tumor). These include those resulting from metastasis, which is the spread of malignant or tumor cells.

[0083] As used herein, the terms "treat," "treatment," and "treating" refer to The progression, severity and progression of a disorder, e.g., a proliferative disorder, resulting from the administration of one or more therapies. and / or reduction or amelioration of the duration or one or more symptoms of the disorder (preferably one or more In a specific embodiment, the term "treat" refers to an improvement in a patient's condition (a discernible symptom of the condition). "Placing" and "treating" refer to conditions that may not always be apparent to the patient, such as tumor growth. refers to the improvement of at least one measurable physical parameter of a proliferative disorder. In this embodiment, the terms "treat", "treatment" and "treating" can be used to refer to, for example, Physical, e.g. by stabilization of symptoms, physiological, e.g. by stabilization of physical parameters, or both. In another embodiment, the term "treat" refers to the inhibition of progression of a proliferative disorder, either of which is a cancer. "Treatment" and "treating" refer to the reduction or stabilization of tumor size or cancerous cell number. .

[0084] The term "subject" as used herein refers to a human. A human may be an adult, an adolescent, a child, or an infant. could be.

[0085] As used herein, the term MDM4 inhibitor refers to an inhibitor that preferentially binds to MDM-4 and inhibits p53 -refers to inhibitors that disrupt MDM4 binding, thus releasing the sequestered p53 protein.

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(2012) Improved detection n suggests all Merkel cell carcinomas ha rbor Merkel polyomavirus.The Journal of clinical investigation 122(12):4645-4653 .10.1172 / JCI64116. 5.Shuda M,Kwun HJ,Feng H,Chang Y,Moore P S(2011)Human Merkel cell polyomavirus sm all t antigen is an oncoprotein targetin g the 4e-bp1 translation regulator.The J ournal of clinical investigation 121(9): 3623-3634.10.1172 / JCI46323. 6.Cheng J,Rozenblatt-Rosen O,Paulson KG, Nghiem P,DeCaprio JA(2013)Merkel cell po lyomavirus large t antigen has growth-pr omoting and inhibitory activities.Journa l of virology 87(11):6118-6126.10.1128 / J VI.00385-13. 7.Starrett GJ et al.(2017)Merkel cell po lyomavirus exhibits dominant control of the tumor genome and transcriptome in vi rus-associated Merkel cell carcinoma.mBi o 8(1).10.1128 / mBio.02079-16. 8.Harms PW et al.(2015)The distinctive m utational spectra of polyomavirus-negati ve Merkel cell carcinoma.Cancer research 75(18):3720-3727.10.1158 / 0008-5472.CAN- 15-0702. 9.Allen MA et al.(2014)Global analysis o f p53-regulated transcription identifies its direct targets and unexpected regul atory mechanisms.eLife 3.10.7554 / elife.0 2200. 10.Bates S et al.(1998)p14ARF links the tumour suppressors RB and p53.Nature 395 (6698):124-5. 11.Nomura K et al.(2017)Structural analy sis of MDM2 RING separates degradation f rom regulation of p53 transcription acti vity.Nat.Struct.Mol.Biol.24(7):578-587. 12.Bista M,Petrovich M,Fersht AR(2013)MD MX contains an autoinhibitory sequence e lement.Proc.Natl.Acad.Sci.U.S.A.110(44): 17814-9. 13.Chen L et al.(2015)Autoinhibition of MDMX by intramolecular p53 mimicry.Proc. Natl.Acad.Sci.U.S.A.112(15):4624-9. 14.Beliveau A,Yaswen P(2007)Soothing the watchman:telomerase reduces the p53-dep endent cellular stress response.Cell cyc le(Georgetown,Tex.)6(11):1284-1287.10.41 61 / cc.6.11.4298. 15.Cheng J et al.(2017)Merkel cell polyo mavirus recruits mycl to the ep400 compl ex to promote oncogenesis.PLOS Pathogens 13(10):1-31. 16.Arora R et al.(2012)Survivin is a the rapeutic target in merkel cell carcinoma .Sci Transl Med 4(133):133ra56. 17.Berrios C et al.(2016)Merkel cell pol yomavirus small t antigen promotes pro-g lycolytic metabolic perturbations requir ed for transformation.PLoS pathogens 12( 11).10.1371 / journal.ppat.1006020. 18.Lenos K et al.(2012)Alternate splicin g of the p53 inhibitor hdmx offers a sup erior prognostic biomarker than p53 muta tion in human cancer.Cancer Research 72( 16):4074-4084.10.1158 / 0008-5472.CAN-12-0 215. 19.Sholl LM et al.(2016)Institutional im plementation of clinical tumor profiling on an unselected cancer population.JCI Insight 1(19):e87062. 20.Houben R et al.(2013)Mechanisms of p5 3 restriction in merkel cell carcinoma c ells are independent of the merkel cell polyoma virus t antigens.The Journal of investigative dermatology 133(10):2453-2 460.10.1038 / jid.2013.169. 21.Kroenke J et al.(2015)Lenalidomide in duces ubiquitination and degradation of ck1alpha in del(5q)mds.Nature 523(7559): 183-188.10.1038 / nature14610. 22.Petzold G,Fischer ES,Thoma NH(2016)St ructural basis of lenalidomide-induced C K1α degradation by the CRL4(CRBN)ubiquit in ligase.Nature 532(7597):127-30. 23.Spiotto MT et al.(2010)Imaging the un folded protein response in primary tumor s reveals microenvironments with metabol ic variations that predict tumor growth. 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Example

[0087] Materials and Methods Plasmid GFP and T antigen cDNA were purchased from David Root (Addgene plasmid 4 1394) and Eric Campeau (Addgene plasmid 17486) pLIX-402-induced empty or pLenti CMV Bla obtained respectively Gateway (Invitrogen) in st empty vector (w263-1) The sgRNA clone for CK1α (BRDN00011493 15, BRDN0001145680) by John Doench and David Ro ot (Addgene plasmids 76188, 76189). 53-shRNA-941 was cloned into the human brain tissue of Todd Waldman (Addgene Plasmid 25 Obtained from 637).

[0088] Cells and cell culture To generate cell lines stably expressing these constructs, three vector lentiviruses were Using the Lus transduction system, IMR90 human lung fibroblasts, HCT116 colon carcinoma or MKL The MCC cell line was obtained from Masa Shuda (Un iversity of Pittsburgh,PA), Juergen Becke r(Medical University Graz,Austria) and Rola nd Houben(University of Wuerzburg,Germany) 293T, HCT116 and IMR90 cells were obtained from ATCC. Generation of MKL-1 MCC cell lines inducibly expressing shRNA and expression of p53DD, M IMR90 transformation using YCL and hTERT constructs was previously described ( 17 ). IMR90 and human primary foreskin fibroblasts (HFF) were cultured in 15% FBS, antibiotics and MCC cell lines were cultured in DMEM supplemented with non-essential amino acids, 10% FBS and Grown in RPMI supplemented with antibiotics.

[0089] Cell viability assay Nutlin-3 (Cayman Chemical), Lenalidomide (Cayman Chemical) hemical), thalidomide (Santa Cruz Biotechnology ), pomalidomide (Selleck Chemicals), AMG232 (MedCh em Express), RG7388 (manufactured by Aileron Therapeutics) ), HDM201 (Novartis Pharmaceuticals), SC-24 -UR99 (Novartis Pharmaceuticals), cycloheximide (Sigma-Aldrich) and MG132 (Boston Biochem). Reconstituted in MSO and added directly to culture medium. Cell viability was measured using the Kit II (XTT) (Roche) according to the manufacturer's instructions. Efficiency assays were performed and BH3 profiling was performed as described by Pallis et al. (36). The procedure was performed as described in Compusyn and Synergyfinder (28,29 ) was used to carry out synergy studies.

[0090] Immunoprecipitation, immunoblotting and antibodies. Confluent cultures of cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated for 1 h. BC lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1:10,000 mercaptoethanol, 0.5 mM EDTA) The lysates were resuspended in 10 ml of water on ice for 10 minutes and then centrifuged. The clarified lysates were incubated with antibodies and magnetic tandem. Protein A / G beads (PureProteome magnetic beads, EMD Millipor e) overnight. The beads were incubated with high salt EBC buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) The gel was washed five times with Laemmli sample buffer before SDS-polyacrylamide gel electrophoresis. Immunoprecipitation and Western blotting were performed using MDM4 (17914-1 -AP;Proteintech Group), MDM2(SMP14, Santa Cruz biotechnology), CK1α(17125-1-AP; Prot eintech Group), Phospho-Ser166MDM2(]3521, Cell Signaling Technology), Phospho-Ser15p53(]928 4; Cell Signaling Technology), Acetyl-Lys382 p53(]2525;Cell Signaling Technology), p21 (]2946;Cell Signaling Technology), PUMA(] 12450;Cell Signaling Technology), VPS39(a b107570;Abcam), PARP(]9542;Cell Signaling Technology), GFP(]2555;Cell Signaling Te chnology), MYCL(14584-1-AP; Proteintech Gr oup), p53(DO-1;Thermo Scientific / Lab Visi on) and RB1 (G3-245; BD Biosciences) were used. The mouse monoclonal antibodies Ab3 and Ab5 against MCV T antigen were used to It is produced against residues 1-260 of the V large T antigen and reacts with glutathione S-transferase in bacteria. The enzyme was produced as a GST fusion protein (17). Quantitative Western blotting was performed using a Li-Cor Biotechnology system. We conducted a survey.

[0091] ChIP- and RT-qPCR The ChIP method was modified from the protocol described by Schmidt et al. (37). Dual cross-linking using disuccinimidyl glutarate (DSG) and formaldehyde MKL-1 cells or IMR90 cells were crosslinked using SimpleCh. Cells were lysed using IP buffer A and B (Cell signaling) and DNA A was used as a micrococcal nuclease (New England Biolabs Inc. ) for 30 min at 37°C, followed by five 20-second cycles of For RT-qPCR, RNeasy Plus Mini was used. Total RNA was purified using the High-Capacity cDNA Kit (Qiagen). cDNA was synthesized using a DNA reverse transcription kit (Applied Biosystems). Brilliant III Ultra-Fast SYBR Green qPCR Master Mix (Agilent Technologies) was used. Quantitative PCR was performed using primers 100-fold higher than those used in the control study. Primer information can be found in Supplementary Table 2.

[0092] Xenograft Efficacy Test 7-9 week old NSG mice (Jackson laboratory) were incubated with 1.00e+ 007 MKL-1 MCC cells were injected. When the tumor reached a size of 200 mm3, Groups of eight mice were treated with vehicle, HDM201 (Novartis Pharma 40 mg / kg in 50 mM phosphate buffer, pH 6.8, 0.5% Methylcellulose (400cP), oral), lenalidomide (MedChemExpres s, 50 mg / kg, 0.5% CMC + 0.25% Tween 80, p.o.) or H Treatment was with both DM201 and lenalidomide daily. The test was terminated when the maximum acceptable size was reached.

[0093] Some of the experiments carried out and the results obtained are described in the Detailed Description of the Invention section and in the These are disclosed herein in the drawings and description.

[0094] Example 1 The following table shows the efficacy of various combinations of drugs / compounds in reducing the viability of MKL-1 MCC cells. We present data from a combination synergy study. A 5-HT-10 antibody was administered to 100 μg / mL of untreated and 100 μg / mL of untreated ... Relative viability (% response) normalized to transplanted cells was determined. Separate tables are shown for combination 1. 1. Nutrin-Lenalidomide 2. Nutrin-UR99 3. HDM201-UR99 4. Nu Torin-3 + / - lenalidomide; RG7388 + / - lenalidomide; lenalidomide alone 5. Includes data on HDM201 + lenalidomide.

[0095] [Table 5]

[0096] [Table 6]

[0097] [Table 7]

[0098] [Table 8]

[0099] [Table 9]

[0100] Incorporation by Reference All publications, patents and accession numbers referred to herein are incorporated by reference in their entirety. All such patents are expressly and individually indicated to be incorporated by reference herein. No. 6,313,992, which is incorporated by reference in its entirety.

[0101] Equivalent While specific embodiments of the present invention have been discussed, the above specification is illustrative. It is to be understood by those skilled in the art that, upon review of this specification and the claims that follow, Many variations of the present invention will become apparent. The full scope of the present invention is to be understood as including all equivalents thereof. By reference to the claims in their entirety and the specification in their entirety, including such variations, The decision must be made based on the above.

Claims

1. A pharmaceutical composition comprising (a) a mouse double minute 2 (MDM2) inhibitor and (b) a casein kinase 1 alpha (CK1α) degrader and / or a mouse double minute 4 (MDM4) inhibitor.

2. 2. The pharmaceutical composition of claim 1, wherein the MDM2 inhibitor is Nutlin-3, Idasanutlin (RG7388), AMG232, or HDM201, i.e., (S)-5-(5-chloro-1-methyl-2-oxo-1,2-dihydro-pyridin-3-yl)-6-(4-chloro-phenyl)-2-(2,4-dimethoxy-pyrimidin-5-yl)-1-isopropyl-5,6-dihydro-1H-pyrrolo[3,4-d]imidazol-4-one.

3. The pharmaceutical composition according to claim 1 or 2, wherein the CK1α degrader and / or MDM4 inhibitor is pomalidomide, lenalidomide, i.e., (RS)-3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione, or SC-24-UR99, i.e., 4-(3-amino-1-(4-chloro-5-methyl-6-(methylamino)pyridin-3-yl)-5-fluoro-1H-indazol-6-yl)naphthalen-1-ol.

4. A pharmaceutical composition described in any one of claims 1 to 3 for use in treating a p53 wild-type (WT) tumor in a subject.

5. Use of a pharmaceutical composition described in any one of claims 1 to 3 for the manufacture of a medicament for the treatment of a p53WT tumor in a subject.

6. 6. The pharmaceutical composition of claim 4 or the use of claim 5, wherein the p53WT tumor is a solid tumor.

7. 7. The pharmaceutical composition of claim 6 or the use of claim 6, wherein the solid tumor is selected from the group consisting of sarcoma, such as liposarcoma or soft tissue sarcoma, lymphoma, such as non-Hodgkin's lymphoma (NHL), in particular mantle cell lymphoma (MCL), melanoma, such as cutaneous melanoma or uveal melanoma, blastoma (e.g. neuroblastoma), colon tumor, colorectal tumor, renal tumor, liver tumor and skin cancer, such as Merkel cell carcinoma (MCC).

8. the solid tumor is Merkel cell carcinoma (MCC), and optionally The pharmaceutical composition according to claim 6 or the use according to claim 6, wherein the Merkel cell carcinoma (MCC) is a Merkel cell polyomavirus (MCV)-positive MCC.

9. 6. The pharmaceutical composition of claim 4 or the use of claim 5, wherein the p53WT tumor is a hematological tumor (or hematological malignancy).

10. the hematological tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL), and optionally 10. The pharmaceutical composition of claim 9 or the use of claim 9, wherein the hematological tumor is myelodysplastic syndrome (MDS).

11. A pharmaceutical composition according to any one of claims 1 to 4 and 6 to 10 or a use according to any one of claims 5 to 10, further comprising one or more further anti-cancer agents.

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