Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with　5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

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Alox-5 As a Potent Therapeutic Target on Overcoming TKI-Resistance in Chronic Myeloid Leukemia with T315I Mutation in Bcr-Abl

Blood ◽

10.1182/blood.v126.23.4835.4835 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4835-4835 ◽

Cited By ~ 1

Author(s):

Jishi Wang ◽

Dan Ma ◽

Ping Wang ◽

Weibing Wu ◽

Lu Cao ◽

...

Keyword(s):

Stem Cells ◽

Cell Lines ◽

Therapeutic Target ◽

Kinase Inhibitors ◽

Mrna Level ◽

Resistant Cell ◽

Tki Resistance ◽

T315i Mutation ◽

Pkc Β

Abstract Background&Significance: Chronic myeloid leukemia(CML) is a malignant disease of a primitive haematological cell, characterised by inappropriate expansion of myeloid cells. Although the disease is readily controlled by Tyrosine kinase inhibitors, approximately one third of patients will eventually fail treatment. And we believed it corresponds to insensitive leukemia stem cells(LSCs) with unresponsive genes to the kinase inhibitors in CML and mutation of Bcr-Abl. 5-lipoxygenase gene(Alox5) was proved as a novel therapeutic target in cancer stem cells of CML. It encodes a member of the lipoxygenase gene family and plays a role in the synthesis of leukotrienes from arachidonic acid. Without Alox5, Bcr-Abl fails to induce CML in mice due to the impairments of the functions of LSCs. However, recent report suggest that Alox5 regulation may not be important for the development of CML in human. Interestingly, we found specific upregulation of Alox5 in CML patients with strongly positive expression of p210 in mRNA level, including the patients primary diagnosed as CML and who suffered in relapse for TKI resistance. Therefore, we characterized the function and regulation of Alox5 in TKI-resistant CML. Results: Firstly, we verified the upregulation of Alox5 by real-time PCR on sorted human CML progenitor populations with strongly positive expression of Bcr-Abl(p210), but not on CML patients obtained remission after treatment of TKI. To evaluate the function and regulation of Alox5, we silenced Alox5 by siRNA and chemical inhibitior in human CML cell lines K562, its TKI-resistant cell lines K562R, murine CML cell lines BaF3wild, and its TKI-resistant cell line BaF3T315I. As a result, the apoptotic rate induced by Alox5 inhibition alone in K562 and BaF3wild cells was lower than by TKI. Conversely, blockage of Alox5 in BaF3T315I cells caused increasingly apoptotic rate, which was higher than imatinib treatment. To further study if Alox5 could play an important role in impairing leukemia stem cells in CML, we cultured LSCs(Lin-c-Kit+Sca-1+) derived from CML patients with Bcr-Abl-T315I mutation in the presence of Alox5 inhibitor or imatinib alone. For 24h treatment, obvious apoptosis was observed in cells cultured with Zileuton, a kind of Alox5 inhibitor, but not in cells cultured with imatinib. Therefore, apoptosis related genes were detected, significant downregulation of Bcl-2 was found compared to in LSCs without Alox5 knockouted. In addition, downregulation of Alox5 followed Bcr-Abl inhibition in CML, we hypothesised that Alox5 was a downstream of Bcr-Abl, genomic array was used to uncover the signaling pathway connected Alox5 with Bcr-Abl. The results shown us that PKC-β was significantly downregulated when Bcr-Abl was inactivated by TKI. Selectivly inhibited PKC-β could decreased Alox5 expression in mRNA level. As for this mechanism, we found p38/MAPK signaling pathway mediated regulation of Alox5 by PKC-β. Next, we evaluated the in vivo anti-CML effect of Alox5 inhibition with a xenograft mice model. Two weeks after the transplantation of human CML cells with mutant Bcr-Abl-T315I(n=12). NOD/SCID/IL2Rg-KO(KOG) mice were treated with normal saline, 10mg/kg Zileuton or imatinib intraperitoneally on everyday schedule. At first, we confirmed that Zileuton doesn't affect normal hematopoiesis(n=3), but eventually inhibite LSCs growth(n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ CML cells and LSCs were significantly reduced by Zileuton treatment in bone marrow((BM) of the recipient mice compared with normal saline-treated control mice and imatinib treated mice, indicating that Alox5 inhibition can inhibit the survival of CML-T315I mutant cells and LSCs in vivo. Conclusion: Together, these results suggest that Alox5 would be a potent therapeutic target on overcoming TKI-resistance involved in Bcr-Abl-T315I mutation. Disclosures No relevant conflicts of interest to declare.

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ASLAN003, a Novel and Potent Dihydroorotate Dehydrogenase (DHODH) Inhibitor, Induces Differentiation of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114392 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4047-4047 ◽

Cited By ~ 1

Author(s):

Jianbiao Zhou ◽

Jessie Yiying Quah ◽

Jing Yuan Chooi ◽

Sabrina Hui-Min Toh ◽

Yvonne Ng ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Research Funding ◽

Tissue Bank ◽

Dihydroorotate Dehydrogenase ◽

Employment Equity ◽

Healthy Control ◽

Equity Ownership ◽

Nbt Reduction

Abstract Background: Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases. Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway. DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML. ASLAN003 is a novel, potent small molecule DHODH inhibitor being developed in AML by ASLAN Pharmaceuticals. Methods: We investigated activity of ASLAN003 in AML cell lines and primary bone marrow (BM) cells (NUS Leukemia Tissue Bank) from patients with AML (N = 14) or myelodysplastic syndromes (MDS) (N = 6) and healthy control (N = 1). We performed CTG assay, FACS analysis of cell viability and myeloid markers, wright-giemsa staining, NBT reduction assay, and qRT-PCR analysis of key lineage transcription factors to evaluate the effects of ASLAN003 on cell growth, differentiation, apoptosis, and gene expression changes in vitro. Two AML cell lines and 1 leukemic patient derived xenograft (PDX) line (NUS Leukemia Tissue Bank) were studied in NSG xenograft mice. Mice were administrated with vehicle control or ASLAN003 50 mg/kg by oral gavage once daily. Results: ASLAN003 inhibited leukemic cell growth of THP-1, MOLM-14 and KG-1 with IC50 of 152, 582 and 382 nM, respectively, at 48 h. Treatment of these leukemia cells with ASLAN003 for 96 h consistently resulted in remarkable increase of CD11b (p < 0.001) and displayed morphologic changes of terminal differentiation and positivity for NBT reduction. ASLAN003 was active in differentiation with an EC50 of 28, 85, and 56 nM, in these 3 lines, respectively. ASLAN003 induced approximately 2-fold higher CD11b+ cells than Brequinar (BRQ), another DHODH inhibitor. Addition of uridine rescued differentiation and improved cell viability in ASLAN003 treated-cells, implying on-target specificity of ASLAN003. Mechanistically, ASLAN003 induced differentiation through induction of myeloid lineage transcription factor Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1. The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate: ≥ 5%, but < 15%; resistant: < 5%. Among AML samples, we observed 6 (43%) sensitive cases, 6 (43%) moderate cases and 2 (14%) resistant cases. Three (50%) MDS samples displayed sensitive response and 3 cases (50%) showed moderate response. The healthy control sample was resistant to ASLAN003. Importantly, ASLAN003 promoted differentiation and cell death of myeloid cells in one relapsed AML case. Morphologic analysis and NBT assay demonstrated the features of neutrophil differentiation in selected ASLAN003-treated primary AML blasts. For in vivo experiments, significantly prolonged survival was seen in ASLAN003-treated groups when compared to vehicle control group in both MOLM-14 (p = 0.031) and THP-1 (p < 0.001) xenograft models. ASLAN003 substantially reduced disseminated tumors and leukemic infiltration into liver in xenografted mice. The human CD45+ cells were significantly reduced in BM, peripheral blood, spleen and liver, with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models (p < 0.01). We also evaluated the therapeutic efficacy of ASLAN003 in one PDX line, AML-14. At the end of experiments (day 77 post treatment), all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs (p = 0.04). Overall, these data demonstrate potent in vivo efficacy of ASLAN003 in inducing myeloid differentiation of blast cells and the drug appears highly tolerable even after prolonged administration. Conclusion: ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation, inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell lines and reduces leukemic burden in an AML PDX model. Currently, ASLAN003 efficacy is being evaluated in a Phase IIa clinical trial in patients with AML (NCT03451084; Ting, ASH abstract 2018). Disclosures Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi:ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng:Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Merck: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

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MPL Overexpression Induces a High Level of Mutant-Calr/MPL Complex: A Novel Mechanism of Ruxolitinib Resistance in Myeloproliferative Neoplasms with Calr Mutations

Blood ◽

10.1182/blood-2020-139162 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 16-17

Author(s):

Shunichiro Yasuda ◽

Satoru Aoyama ◽

Ryoto Yoshimoto ◽

Daisuke Watanabe ◽

Hiroki Akiyama ◽

...

Keyword(s):

Tumor Cells ◽

Cell Lines ◽

Immunohistochemical Staining ◽

Research Funding ◽

Resistant Cell ◽

Calr Mutations ◽

Immunocompromised Mice ◽

High Level ◽

Resistant Cells

【Introduction】 Although Ruxolitinib (RUX), a JAK1/2-inhibitor, is an effective treatment option for primary myelofibrosis, tumor cells become resistant to this drug in many MPN patients, causing poor prognosis of MPN patients. Until now, various studies have elucidated the mechanisms of RUX-resistance in JAK2V617F-mutant MPN cells, including 1) increased JAK2 heterodimerization leading to sustained JAK2 activation, 2) JAK2 kinase domain mutations, and 3) JAK1/JAK3 activations. However, mechanisms of RUX-resistance in MPN cells with CALR mutations have not been fully characterized to date. In this study, we have clarified a mechanism of RUX-resistance in MPN tumor cells with CALR mutations. 【Materials and Methods】 At first, we have created several human cell lines with exogenous MPL expressions (exMPL) and CALR +1 frameshift mutations (CALR-fs) by introductions of V5-tagged MPL and/or FLAG-tagged CALR Del52/Ins5 expressing vectors or the CRISPR/Cas9 technology. We have confirmed that these cell lines had increased JAK/STAT signaling and respond to RUX-treatment. To establish RUX-resistant cell lines, we have cultured these cell lines with low-dose RUX (0.2μM), and gradually increased the concentrations of RUX by 0.1μM every week. We have successfully established RUX-resistant cells that proliferated in the presence of RUX at 0.8μM. Then, we characterized the RUX-resistant cells with CALR-fs/exMPL. To examine whether RUX-resistant cells shows the resistant-phenotype in vivo, we have subcutaneously implanted RUX-resistant cells as well as RUX-sensitive cells into immunocompromised mice. Three weeks after injections of the tumor cells, the mice were euthanized, and the subcutaneous tumors pathologically examined. MPL knockdown experiments showed that high levels of MPL were indispensable in the resistant cell lines. Co-immunoprecipitation assaies showed the interactions of mutant CALR and MPL proteins in RUX-resistant cells. To examine reversibility of RUX-resistance, RUX-resistant cells were cultured without RUX for three months. Finally, we examined pathological features of bone marrow samples of MPN patients with CALR mutations by immunohistochemical staining. 【Results】We have found that RUX-resistant cells had high MPL transcripts, overexpression of both immature and mature MPL, and JAK2. We also found that RUX-resistant cells had increased phosphorylations of JAK1, JAK2, JAK3 STAT5, MEK and ERK. In vivo assay using immunocompromised mice showed the immunohistochemical staining of MPL in the tumors from RUX-resistant cells showed high expression of MPL in the tumor cells. We also found that mature MPL proteins were more stable since proteasome-dependent degradation of mature MPL proteins was impaired in RUX-resistant cells. Knockdown of MPL of RUX-resistant cells by shRNAs decreased intensity of phosphorylations of JAK1, JAK2, STAT5, MEK and ERK, suggesting that the high expression of MPL leads to more potent signaling. Notably, when mutant-CALR proteins were immunoprecipitated, both immature and mature MPL proteins were co-immunoprecipitated: more MPL proteins were pulled down in RUX-resistant cells. In a reciprocal experiment, when MPL proteins were immunoprecipitated, mutant-CALR proteins were co-immunoprecipitated: more mutant-CALR proteins were pulled down in RUX-resistant cells. Reduction of mutated CALR decreased proliferation of the resistant cells, suggesting that a high level of mutant-CALR/MPL complex contributed to RUX-resistance in these cells. When the resistant cells were cultured in the absence of RUX, RUX-resistance was reversed with reduction of MPL transcripts, mature MPL and JAK2 proteins and mutant-CALR/MPL complex. Immunohistochemical staining showed that MPL staining intensity of megakaryocytes of MF patients with CALR mutations were higher than those of patients with JAK2V617F or normal individuals. MPL expressions were higher after RUX-treatment in MF patients with both JAK2V617F mutations and CALR mutations. 【Discussion】 Overexpression of MPL is a common mechanism after RUX-treatment in MPN cells with both JAK1V617F mutations and CALR mutations. In RUX-resistant cells with CALR mutations, MPL overexpression leads to the high level of mutant-CALR/MPL complex, causing resistance to RUX. This novel mechanism could be a new therapeutic target to overcome RUX-resistance in MPN cells with CALR mutations. Disclosures Komatsu: Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

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Acid Ceramidase (ASAH1) Mediates Intrinsic and Intercellular Transfer of Proteasome Inhibitor Resistance in Multiple Myeloma

Blood ◽

10.1182/blood-2021-152686 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1206-1206

Author(s):

Ryan T Bishop ◽

Tao Li ◽

Raghunandan R Alugubelli ◽

Oliver Hampton ◽

Ariosto Siqueira Silva ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Resistant Cell ◽

Clinical Samples ◽

In Vivo Model ◽

Acid Ceramidase ◽

Advisory Committees

Abstract INTRODUCTION: Despite proteasome inhibitors (PIs) improving multiple MM (MM) outcomes, patients often become resistant. Identifying mechanisms of resistance with translational potential are an urgent unmet clinical need. Preliminary studies from our group have identified that the therapeutically targetable acid ceramidase, ASAH1, is a key mediator of PI resistance and its presence in extracellular vesicles (EVs) derived from resistant MM cells, confers PI resistance on drug naïve MM cells. METHODS: Nanosight technology, transmission electron microscopy and immunoblot were used to define EVs. Viability and apoptosis assays were used to determine the effects of EVs and inhibitors on resistance acquisition/sensitization to PIs. LC-MS was used to interrogate EV cargo contents. Clinical relevance of ASAH1 was determined in multiple human data cohorts (M2GEN and MMRF CoMMpass). Genetic (shRNA) and pharmacological (ceranib-2) approaches were used to assess the role of ASAH1 mechanistically in vitro and in vivo using multiple isogenic naïve and PI resistant cell lines, patient derived CD138+ MM cells and NSG mouse models. RESULTS: Co-culture of sensitive MM cells with resistant MM-EVs alone significantly protected against PI cytotoxicity. Proteomic profiling revealed high levels of ASAH1 in EVs derived from PI resistant MM cells. Further, we observed ASAH1 is abundant in lysates of multiple PI resistant cell lines compared to their isogenic drug sensitive counterparts. In human datasets, high ASAH1 expression was noted in PI resistant MM patients compared to those newly diagnosed and correlated with significantly shorter survival times. Mechanistically, knockdown of ASAH1 led to reduced conversion of ceramide to sphingosine 1-phosphate (S1-P) and decreased expression/activity of the anti-apoptotic proteins MCL-1, BCL2 and BCL-xL and increases in pro-apoptotic BIM and NOXA. Notably, ASAH1 knockdown also significantly sensitized the cells to PI treatment and this effect was rescued by addition of exogenous S1-P. Pharmacological inhibition of ASAH1 with ceranib-2 also sensitized resistant cells to PI treatment and prevented EV mediated resistance transfer in vitro. This was recapitulated ex vivo with human clinical samples. Our orthotopic in vivo model using PI-resistant U266-PSR cells show that ceranib-2 is highly effective in limiting the growth of PI-resistant disease, protecting against MM induced bone disease, and increasing overall survival compared to both bortezomib and vehicle controls. CONCLUSION: We define the ceramidase ASAH1 as a novel, druggable target for the treatment of PI resistant MM. Disclosures Hampton: M2Gen: Current Employment. Siqueira Silva: AbbVie Inc.: Research Funding; Karyopharm Therapeutics Inc.: Research Funding. Shain: Janssen oncology: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi Genzyme: Consultancy, Speakers Bureau; Karyopharm Therapeutics Inc.: Honoraria, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; GlaxoSmithLine, LLC: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies Corporation: Consultancy, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Combination Therapy of Bcl-2/X L dual Inhibitor AZD0466 with Acalabrutinib to Overcome Therapeutic Resistance in Aggressive R/R Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-151609 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1867-1867

Author(s):

Yijing Li ◽

Yang Liu ◽

Yuxuan Che ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

...

Keyword(s):

Combination Therapy ◽

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Single Agent ◽

Dual Inhibitor ◽

Car T ◽

Mantle Cell

Abstract Introduction As a rare form of non-Hodgkin's lymphoma, mantle cell lymphoma (MCL) is an aggressive subtype. This is largely due to frequent relapses after therapies including paradigm shifting therapies BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, and Bcl-2 inhibitor (Bcl-2i) venetoclax after long-term treatment in the clinic. Dysregulation of Bcl-2 and Bcl-X L, contributes to therapeutic resistance in MCL. AZD0466 is a novel and highly potent Bcl-2/X L dual inhibitor with active moiety AZD4320. Our preliminary data showed AZD4320 is potent in inhibiting cell viability of MCL cells (IC 50 = 1.6-78 nM). In this study, we assessed the combination efficacy of AZD4320/AZD0466 and acalabrutinib on preclinical MCL models. Methods Cell viability assay was performed to assess the in vitro efficacy of AZD4320 and acalabrutinib alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD4320 and acalabrutinib enhanced cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by immunoblotting. Since AZD4320 is limited in preclinical model due to physicochemical properties and dose limiting cardiovascular toxicity, AZD0466, the drug-dendrimer conjugate of AZD4320, was used for in vivo experiment. In vivo efficacy of AZD0466 (34 mg/kg, weekly, iv) and acalabrutinib (20 mg/kg, BID, oral) alone or in combination was evaluated using a Mino-venetoclax-R (Mino-R) cell xenograft model and a PDX model derived from an ibrutinib-CAR-T dual-resistant MCL patient. Results AZD4320 in combo with acalabrutinib inhibited cell proliferation synergistically in both ibrutinib/venetoclax-sensitive and -resistant cell lines (combination index = 0.17-0.93). Compared to vehicle or either single agent, the combination enhanced cell apoptosis by increasing pro-apoptotic markers cleaved caspase 3 and cleaved PARP. In the xenograft mouse model derived from venetoclax-resistant Mino-R cells, co-treatment of AZD0466 and acalabrutinib decreased tumor size significantly compared to vehicle (n = 5, p < 0.0001) or either single agent (n = 5, p = 0.0118 and 0.0070, respectively). Furthermore, in the PDX mouse model derived from a patient relapsed subsequently from ibrutinib and CAR T therapy, the combination of AZD0466 and acalabrutinib inhibited tumor growth compared to vehicle or either single agent. Acalabrutinib or AZD0466 improved survival compared with vehicle by 14 days or 32 days, respectively. Compared to Acalabrutinib or AZD0466, the combination therapy extended survival by 25 days and 7 days, respectively. All mice tolerated the treatment dose without any weight loss compared to the vehicle or either single agent group. Conclusion Compared to AZD4320/AZD0466 and acalabrutinib, combination therapy demonstrated anti-MCL synergy both in vitro and in vivo. These findings suggest that targeting Bcl-2/X L and BTK is promising to overcome multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang: DTRM Biopharma (Cayman) Limited: Consultancy; BeiGene: Consultancy, Honoraria, Research Funding; Physicians Education Resources (PER): Honoraria; Anticancer Association: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; CAHON: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Epizyme: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; BGICS: Honoraria; Imedex: Honoraria; Clinical Care Options: Honoraria; Celgene: Research Funding; Genentech: Consultancy; Loxo Oncology: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Molecular Templates: Research Funding; Lilly: Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Oncternal: Consultancy, Research Funding; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Scripps: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Chinese Medical Association: Honoraria; Pharmacyclics: Consultancy, Research Funding; Juno: Consultancy, Research Funding; CStone: Consultancy; Bayer Healthcare: Consultancy; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Dava Oncology: Honoraria; Moffit Cancer Center: Honoraria; Mumbai Hematology Group: Honoraria.

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AZD4320 Is a Novel and Potent BCL-2/XL Dual Inhibitor in Targeting Aggressive Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2020-140775 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 44-44

Author(s):

Yijing Li ◽

Yang Liu ◽

Joseph McIntosh ◽

Alexa A Jordan ◽

Angela Leeming ◽

...

Keyword(s):

Cell Lines ◽

Cell Apoptosis ◽

Research Funding ◽

Cell Lymphoma ◽

Publicly Traded ◽

Dual Inhibitor ◽

Car T ◽

Mantle Cell

Introduction: Mantle cell lymphoma (MCL) is a rare subtype of B-cell non-Hodgkin's lymphoma. It is an incurable disease with frequent relapse from chemotherapies, targeted therapies, and cell therapies. Dysregulated expression of BCL-2 family members resulting in enhanced cell survival frequently occurs in many cancer types and often contributes to the development of therapeutic resistance. The BCL-2 inhibitor venetoclax has been shown to be effective in treating refractory/relapsed MCL patients. However, resistance often occurs and one of the underlying mechanisms of this resistance is the increased expression of other anti-apoptotic BCL-2 family members, such as BCL-XL and MCL-1. In this study, we assessed the in vitro and in vivo efficacy of a novel and highly potent BCL-2/XL dual inhibitor AZD4320 in preclinical models. Methods: Cell viability assay was tested after 72-hour treatment with AZD4320 in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines by CellTiter-Glo (Promega). The assay was also done after a 24-hour treatment in primary PDX cells. Cell apoptosis assay was performed to determine if AZD4320 induces cell apoptosis in MCL cell lines. Furthermore, the in vivo efficacy of AZD4320 was assessed in a CAR-T resistant MCL patient-derived xenograft (PDX) model. Results: AZD4320 significantly inhibited cell proliferation in all tested MCL cell lines, including both ibrutinib/venetoclax-sensitive and -resistant cell lines. It had an IC50 value at a low nanomolar range between 0.59 nM to 18 nM. Consistently, AZD4320 was effective in targeting primary PDX cells ex vivo. AZD4320 induced cell apoptosis in a dose-dependent manner. AZD0466, the nanomedicine formulation of AZD4320 (30mg/kg, weekly, IV), dramatically inhibited tumor growth and prolonged mouse survival in an ibrutinib-CAR-T dual-resistant PDX mouse model. All mice tolerated the treatment dose without any body weight loss. Conclusion: The novel BCL-2/XL dual inhibitor AZD4320 demonstrated excellent anti-MCL activity in both ibrutinib/venetoclax-sensitive and -resistant MCL cells in vitro. This was further validated in vivo in a ibrutinib-CAR-T dual-resistant PDX model. These findings provide evidence that dual targeting of BCL-2 and BCL-XL by AZD4320 is promising as it may overcome therapeutic resistance in relapsed/refractory MCL. Disclosures Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado:AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Wang:OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Loxo Oncology: Consultancy, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Lu Daopei Medical Group: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; Pulse Biosciences: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Beijing Medical Award Foundation: Honoraria; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Guidepoint Global: Consultancy; Targeted Oncology: Honoraria; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy.

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Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) Inhibits Myeloma in Vivo.

Blood ◽

10.1182/blood.v104.11.2401.2401 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2401-2401

Author(s):

Makoto Hamasaki ◽

Teru Hideshima ◽

Pierfrancesco Tassone ◽

Paola Neli ◽

Kenji Ishitsuka ◽

...

Keyword(s):

Growth Factor ◽

Cell Growth ◽

Cell Lines ◽

Cell Apoptosis ◽

Parp Cleavage ◽

Dose Dependent Fashion ◽

Orally Bioavailable ◽

Induced Apoptosis ◽

Endothelial Growth Factor

Abstract Atiprimod (N-N-diethl-8,8-dipropyl-2-azaspiro [4.5] decane-2-propanamine) is an orally-bioavailable cationic amphiphilic compound which significantly inhibits inflammation in rat arthritis and multiple sclerosis models. It acts, at least in part, by significantly inhibiting production of interleukin (IL)-6. Since IL-6 mediates MM cell growth, survival and drug resistance in the bone marrow (BM) microenvironment, we in this study characterized the effect of Atiprimod on human MM cells. We first demonstrated that Atiprimod significantly inhibited growth (p<0.05) in MM.1S, U266, and RPMI8226 MM cell lines in a time- and dose-dependent fashion, with IC50s of 0.5–1.25 mM. It also induced cytotoxicity in dexamethasone (Dex)-, doxorubicin (Dox)-, and melphalan (Mel)-resistant MM cell lines; as well as in patient MM cells, but not in normal PBMCs. Atiprimod triggered MM cell apoptosis via activation of caspase-8 and caspase-3, followed by PARP cleavage. Importantly, neither IL-6 nor insulin-like growth factor (IGF)-1, which completely abrogate Dex-induced apoptosis, protects against Atiprimod-induced apoptosis in MM.1S cells. In combination treatment studies, both conventional (Dex, Dox, Mel) and novel (As2O3) agents augment MM cell apoptosis induced by Atiprimod. Atiprimod inhibits STAT3 and Akt, but not ERK1/2, phosphorylation triggered by IL-6, BAX, Bcl-xl, and Mcl-1 in MM.1S cells. Importantly Atiprimod inhibits both IL-6 and vascular endothelial growth factor (VEGF) secretion in BM stromal cells (BMSCs) triggered by MM cell adherence to BMSCs, as well as associated MM cell growth. Finally, Atiprimod inhibits tumor OPM1 MM cell growth in vivo and prolongs survival in a SCID mouse model. Our data therefore demonstrate that Atiprimod both induces MM cell apoptosis and inhibits cytokine secretion in the BM milieu, providing the framework for ongoing clinical trials of this agent to improve patient outcome in MM.

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Dysfunctional HDAC8 Impacts Genomic Integrity and Is a Novel Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood-2021-153382 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1610-1610

Author(s):

Zuzana Chyra ◽

Srikanth Talluri ◽

Rao Prabhala ◽

Mehmet K. Samur ◽

Anil Aktas-Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Hdac Inhibitors ◽

Dependent Manner ◽

Dna Breaks ◽

Advisory Committees

Abstract The histone modifications and associated changes in chromatin structure and function have emerged as important epigenetic mechanisms impacting gene expression and have significant translational relevance in cancers, including multiple myeloma (MM). Epigenetic intervention with histone deacetylases (HDACs) inhibitors is emerging as a promising therapeutic strategy in combination with current anti-myeloma agents. Although pan-HDAC inhibitors have been shown to be effective both in preclinical and clinical setting, they seem to be associated with toxicity. It is, therefore, extremely important to understand the biological and molecular roles of individual HDACs to then selectively target them to limit toxicities observed with pan-HDAC inhibitors. Based on our observation that elevated HDAC8 expression correlates with poor overall survival in MM patients in three different datasets including one publicly available dataset (GSE39754), we evaluated its functional role in MM. HDAC8, a member of class I HDAC isoenzymes, is responsible for the deacetylation of lysine residues on the N-terminal part of the core histones as well as non-histone proteins. We performed genetic modulation of HDAC8 by loss-of-function studies, using shRNA as well as siRNAs targeting HDAC8. Downregulation of HDAC8 in 3 different MM cell lines caused MM cell growth inhibition in a time-dependent manner which was associated with induction of cell apoptosis. Consistently, treatment with a selective and potent HDAC8 inhibitor (OJI-1) caused a significant inhibition of MM cell growth in a panel of 20 MM cell lines (IC50 = 80 nM) in a time- and dose-dependent manner, while having a minimal impact on six PBMC samples from healthy donors both in resting and activated state (IC50 = 150 nM). The mechanism of cell death was apoptosis as demonstrated by annexin-labeling. Importantly, both the HDAC8 knockdown and OJI-1 treatment inhibited DNA breaks as evidenced from γH2AX expression or a single cell gel electrophoresis method to visualize and quantitate DNA breaks. HDAC8 inhibition also caused inhibition of RAD51 foci and HR activity, as measured by strand-exchange assay. Interestingly, non-homologous end joining in MM cells was not impacted by these treatments. Consistent with these data, the overexpression of HDAC8 in MM as well as in normal cells increased DNA breaks and HR activity. Furthermore, the inhibition of HDAC8 (by knockdown and OJI-1) inhibited, whereas its overexpression increased genomic instability, as assessed by micronucleus assay, in surviving MM cells. We also demonstrate that HDAC8 interacts with RAD51 and impacts its acetylation. The treatment of MM cells with HDAC8 inhibitor (OJI-1) increased RAD51 acetylation. Next, we examined the in vivo efficacy of the HDAC8 conditional knockdown in a human xenograft mouse model, using H929 cells injected subcutaneously in SCID mice. HDAC8 knockdown not only caused a significant reduction in tumor growth but also increased survival (p=0.0016) compared to mice injected with control cells. Evaluation of tumors from these mice confirmed in vivo inhibition of DNA breaks and HR activity, and induction of apoptosis following HDAC8-knockdown. HDAC8 inhibitor OJI-1 also synergistically increased the cytotoxicity of existing MM drugs including dexamethasone, bortezomib and lenalidomide. In conclusion, our results demonstrate that elevated HDAC8 in MM cells is involved in inhibition of apoptosis but also contributes to increased DNA breaks and dysregulation of homologous recombination and genome stability. Therefore, HDAC8 is a novel target for therapeutic application in MM. Selective and potent HDAC8 inhibitor OJI-1 has shown a favorable therapeutic index with synergistic effect in combination with existing MM drugs. Disclosures Hajek: Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding. Munshi: Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Karyopharm: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy; Legend: Consultancy.

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POU2AF1 As a Master Regulator of Oncogenic Transcription Factor Networks in Myeloma

Blood ◽

10.1182/blood-2020-142580 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Ricardo De Matos Simoes ◽

Ryosuke Shirasaki ◽

Huihui Tang ◽

Shizuka Yamano ◽

Benjamin G Barwick ◽

...

Keyword(s):

Cell Growth ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Rna Seq ◽

Advisory Committees ◽

Genome Scale ◽

Crispr Activation

Background: Functional genomics studies based on CRISPR and shRNA have documented that multiple myeloma (MM) cells are preferentially dependent (compared to other neoplasias) on a series of TFs, including IKZF1 and IKZF3 (which are targeted by thalidomide derivatives) and others that are not amenable to degradation or small molecule inhibition. Transcriptional co-factors have been therapeutically targeted, for example, inhibitors of BRD4, a co-factor for pTEFB, can be used to down-regulate c-myc. Aim: To identify new transcriptional vulnerabilities in MM with an emphasis on transcriptional co-factors Methods: We integrated results from genome-scale studies using the AVANA library for loss-of-function by gene editing (in 19 MM lines) and the Calabrese library for CRISPR-mediated gene activation (in 5 MM lines) to identify critical transcriptional co-factors (co-TFs). RNA-Seq analysis was used to identify critical pathways affected by POU2AF1 activation and existing ChIP-Seq tracks in MM cells were reanalyzed. Results: POU2AF1 (OCA-B) was the most preferentially essential TF co-factor in MM cell lines vs. non-MM and one of top genes which, upon CRISPR activation in genome-scale studies, increased MM cell fitness in vitro. We further confirmed the role of this gene using focused libraries of sgRNAs against POU2AF1 in vitro and in an in vivo model of MM cell growth in bone marrow-like scaffolds "functionalized" with humanized mesenchymal bone marrow stromal cells to simulate the human BM. CRISPR activation of POU2AF1 is associated with increased MM cell growth. RNA-Seq of POU2AF1 activation in LP1 cells a transcriptional program characterized by upregulation of other genes that are preferentially essential for MM including PRDM1, SUPT7L, UBE2G2 and TSC1; broad-spectrum oncogenic dependencies (e.g KRAS) and genes known or proposed to be involved in the pathophysiology of MM or other neoplasias (e.g. RUNX2, FGFR3, SMO, CREB5, TNFRSF13B, MEF2D, PCGF2). POU2AF1 overexpression was also associated with down-regulation of CDKN1C; of MHC class II molecules and their transcriptional activator CIITA, suggesting that POU2AF1 activation could also contribute to increased MM growth in vivo by allowing escape from immune surveillance. ATAC-Seq data and genome-wide ChIPseq for H3K27Ac in MM cell lines indicate that chromatin surrounding the POU2AF1 locus was highly accessible, concordant with the consistent expression of this TF in MM cell lines and patient-derived cells. CoMMpass data showed that POU2AF1 expression was enhanced in a subset of MM patients at relapse compared to diagnosis. Motif analysis of ChIP-seq data for POU2AF1 identified significant overlap with motifs for TFs relevant to the POU family (e.g. Oct11, Oct2, Oct4); members of the ETS family (e.g. ELF1, Elf4, GABPA); and other TFs with roles in MM including c-myc; IRF4; NF-kappaB, PRDM1, RUNX2 and the POU2AF1 target CREB5. These data suggest a functional interaction between POU2AF1 and other MM-relevant TFs. The transcriptional signature of POU2AF1 activation is enriched for genes downregulated by suppression/inhibition of MM-preferential TFs or epigenetic regulators including IRF4, PRDM1, IKZF1/3 and DOT1L. POU2AF1 binding motifs are also enriched in the promoter regions of MM-preferential dependencies including several MM-preferential TFs. Conclusions: POU2AF1 is essential for MM cells in vitro and in vivo; has a significantly more pronounced and recurrent role as a dependency in MM compared to most other neoplasias; and can further drive MM cell growth, through its ability to interact with several TFs critical for MM, forming multi-protein functional complexes. These results establish POU2AF1 as a central component in the regulatory network of oncogenic TFs in MM and highlight the value of further exploring POU2AF1 as a therapeutic target in MM. Disclosures Downey-Kopyscinski: Rancho BioSciences, LLC: Current Employment. Tsherniak:Cedilla Therapeutics: Consultancy; Tango Therapeutics: Consultancy. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Mitsiades:FIMECS: Consultancy, Honoraria; Ionis Pharmaceuticals, Inc.: Consultancy, Honoraria; Arch Oncology: Research Funding; Janssen/Johnson & Johnson: Research Funding; Karyopharm: Research Funding; TEVA: Research Funding; Takeda: Other: employment of a relative; Fate Therapeutics: Consultancy, Honoraria; Sanofi: Research Funding; Abbvie: Research Funding; EMD Serono: Research Funding.

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Novel PI3K Inhibitor Compound A Induces Myeloma Cell Apoptosis and Shows Synergistic Cytotoxicity with Dexamethasone In Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.4080.4080 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4080-4080

Author(s):

Yuhuan Zheng ◽

Jing Yang ◽

Liang Zhang ◽

Jianfei Qian ◽

Jairo Matthews ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Growth ◽

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Cell Apoptosis ◽

Compound A

Abstract Abstract 4080 Phosphatidylinositol 3-kinase (PI3K) plays a central role in cell metabolism. PI3K is activated by growth factors, cytokines, and other stimulatory factors in association with their receptors. Activated PI3K in turn initiates signaling transduction to Akt-mTOR and leads to regulation of cell growth, proliferation, and apoptosis. Dysregulation of the pathway is widely seen in different types of human cancers, including multiple myeloma (MM). Therefore, PI3K-Akt inhibition is expected to exert broad anti-MM activity. Compound A (CA) is a novel pan-PI3K inhibitor, developed by Novartis Oncology. This compound has shown significant cell growth inhibition and induction of apoptosis in a variety of tumor cell lines. CA is currently being investigated in Phase I clinical trials in solid tumor patients. In this study, we investigated the in vitro and in vivo anti-MM activity of CA. Our findings showed that CA induces apoptosis in MM cell lines, ARP1, ARK, MM.1S, MM.1R, CAG and U266, and primary MM cells in both a time-dependent and a dose-dependent manner in vitro. Western blot analysis indicated activation of caspases after CA exposure. The presence of MM bone marrow stromal cells (BMSCs) or addition of IL-6, the growth cytokine for MM, did not attenuate CA-induced MM cell apoptosis. More importantly, CA only showed limited cytotoxicity toward normal lymphocytes or non-tumoric BMSCs. Results from mechanistic studies showed that CA treatment results in cell cycle arrest in G1 phase by upregulating cell cycle repressor p27 (Kip1) and downregulating cyclin D1. CA treatment also caused decreased anti-apoptotic XIAP expression, and increased cytotoxic small isoform of Bim, BimS expression, both of which may contribute to CA-induced cell apoptosis. In addition to its effect in vitro, CA showed potent anti-MM activity in vivo in an established MM model in SCID mice. CA treatment repressed tumor growth and prolonged the survival of tumor-bearing mice. To test the synergistic/addictive effect of CA with other MM chemotherapeutics, we combined CA with melphalan, dexamethasone, lenalidomide, or bortezomib to treat MM cells. Our results showed that low doses of CA and dexamethasone, either of which alone has only limited cytotoxicity, exhibited synergistic anti-MM activity in dexamethasone-sensitive cell lines ARP1 and MM.1S, but not in dexamethasone-resistant cell MM.1R. Western blot analysis suggested that CA and dexamethasone combined treatment in MM.1S results in accumulation of the cytotoxic BimS. Increased BimS expression may cause the synergistic effect of CA and dexamethasone. Thus, our findings suggest CA alone or together with dexamethasone may be a promising treatment for MM. Disclosures: No relevant conflicts of interest to declare.

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