Combination of Selinexor and the Proteasome Inhibitor, Bortezomib Shows Synergistic Cytotoxicity in Diffuse Large B-Cells Lymphoma Cells In Vitro and In Vivo

Abstract Background: XPO1 (exportin-1/CRM1) mediates nuclear export of proteins containing leucine-rich amino-acid consensus sequences. XPO1 cargo proteins include many of the major tumor suppressor proteins (p53, IkB, pRB, FOXOs) and their export leads to the inactivation of cell cycle checkpoints. Overexpression of XPO1 has been reported to correlate with poor cancer prognosis. The Selective Inhibitor of Nuclear Export (SINE) compound, selinexor, binds covalently to the cargo pocket on XPO1, inhibits nuclear export which leads to cell cycle arrest and specific cancer cell death. Selinexor is currently in advanced clinical trials for patients with solid and hematological malignancies including patients with relapsed/refractory Diffuse Large B-Cell Lymphoma (DLBCL) (NCT02227251). Using preclinical models, we recently demonstrated that proteasome inhibitors (PI) can re-sensitize multiple myeloma that acquired resistance to selinexor. Here, we aimed to find if treatment with selinexor and bortezomib is beneficial for the treatment of DLBCL. Methods: DLBCLcell lines were treated with selinexor in combination with bortezomib. Cell viability was examined using standard viability assays after 72 hours of treatment. Whole cell protein lysates were evaluated by immunoblotting. NF-κB transcriptional activity was analyzed using an ELISA assay. WSU-DLCL2 cells were grown as sub-cutaneous tumors in ICR SCID mice. Tumor bearing mice were divided into 4 groups and were administered either vehicle, sub-maximum tolerated doses of selinexor (10 mg/kg p.o. twice a week, M, Th), bortezomib (1 mg/kg i.v. twice a week, M, TH) and the combination of selinexor (10 mg/kg p.o. twice a week) plus bortezomib (1 mg/kg i.v. twice a week). Results: The combination treatment of selinexor with bortezomib synergistically killed DLBCL cells compared to the single agents alone. Co-treatment with bortezomib enhanced selinexor mediated nuclear retention of IκB-α. Selinexor plus bortezomib treatment decreased NF-κB transcriptional activity. Finally, the combination of selinexor with bortezomib showed superior anti-tumor efficacy in the combination group compared to single agent treatments in WSU-DLCL2 xenograft model. Conclusions: Based on our results, inhibition of NF-κB transcriptional activity through forced nuclear retention of IκB appears to be an important mechanism underlying the synergistic effects of selinexor plus bortezomib in many different cell lines including DLBCL. The superior efficacy of selinexor plus bortezomib combination both in vitro and in vivo when compared to single agents along provides a rational for conducting clinical trials with these combinations in DLBCL patients. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kauffman:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership.

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In Vitro and in Vivo Quantification of Exportin-1 (XPO1) Occupancy By the Oral Selective Inhibitor of Nuclear Export (SINE) Selinexor in Multiple Myeloma, Acute Myeloid Leukemia, and Healthy PBMCs

Blood ◽

10.1182/blood.v124.21.5196.5196 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5196-5196

Author(s):

Marsha Crochiere ◽

Boris Klebanov ◽

Erkan Baloglu ◽

Ori Kalid ◽

Trinayan Kashyap ◽

...

Keyword(s):

Board Of Directors ◽

Nuclear Export ◽

Drug Exposure ◽

Employment Equity ◽

Advisory Committees ◽

Hel Cells ◽

Equity Ownership ◽

Cargo Binding

Abstract Introduction: SINE are a family of small molecules that selectively inhibit nuclear export by forming a slowly reversible covalent bond with Cysteine 528 (Cys528) in the cargo binding pocket of Exportin 1 (XPO1/CRM1). SINE binding to XPO1 leads to forced nuclear retention and activation of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB, resulting in selective death of cancer cells. Selinexor is an orally bioavailable SINE compound currently in human phase I and II clinical trials for advanced hematological and solid cancers. Oral selinexor demonstrates maximal pharmacokinetic exposure at 1-2 hours in humans with associated increases in pharmacodynamic markers of XPO1 inhibition in 2-4 hours that last for up to 48 hours. The goal of this study was to develop a binding assay that would enable quantification of XPO1 occupancy in PBMCs from patients following oral administration of selinexor. Methods: To measure the binding of SINE to XPO1, biotinylated leptomycin B (LMB) was utilized. Biotinylated LMB binds covalently and irreversibly to Cys528 in the cargo-binding site of free XPO1 with activity confirmed to be similar to that of unmodified LMB in cytotoxicity assays. To measure SINE binding to XPO1 in vitro, cancer cell lines and PBMCs from normal human donors were treated with SINE compounds prior to treatment with biotinylated LMB. Any XPO1 that did not bind SINE instead binds to biotinylated LMB and can be quantified. In in vivo studies, mice were treated with selinexor, followed by collection of PBMCs for treatment with biotinylated LMB. After incubation with biotinylated LMB, cells were harvested, lysed, and protein lysates were subjected to pull-down experiments with streptavidin-conjugated beads followed by immunoanalysis of XPO1. Results: To evaluate selinexor-XPO1 binding kinetics in vitro, MM.1S, AML2, AML3, and HEL cells were treated with 0 - 10 µM of SINE compounds and unbound XPO1 was pulled down from cell lysates treated with biotinylated LMB. Immunoanalysis showed that 50% XPO1 occupancy with selinexor was achieved at 0.07 µM in MM.1S, 0.1 µM in AML2, 0.03 µM in AML3, and 0.12 µM in HEL cells. Selinexor-XPO1 occupancy experiments using human PBMCs isolated from donor whole blood showed 50% XPO1 occupancy at 0.05 µM. In mice, 50% XPO1 occupancy in PMBCs was achieved after 4 hours treatment with 1.2 mg/kg (3.6 mg/m2) selinexor, while 90% XPO1 occupancy was achieved at 8.1 mg/kg (24.3 mg/m2). Mice treated with a single dose of selinexor from 1.5 to 10 mg/kg for 4-96 hours revealed sustained, dose dependent XPO1 occupancy in PBMCs for up to 72 hours. Conclusions: We have developed a sensitive and robust assay to measure selinexor binding to XPO1 that can be used to evaluate drug exposure following treatment with oral selinexor in preclinical and clinical studies. Studies are ongoing to determine whether there is a correlation between XPO1 occupancy (pharmacodynamics measurement) with disease response in patients with solid and hematological malignancies. Disclosures Crochiere: Karyopharm: Employment. Klebanov:Karyopharm Therpeutics: Employment. Baloglu:Karyopharm: Employment. Kalid:Karyopharm Therapeutics: Employment. Kashyap:Karyopharm Therapeutics: Employment. Senapedis:Karyopharm: Employment. del Alamo:Karyopharm: Employment. Rashal:Karyopharm Therapeutics: Employment. Tamir:Karyopharm: Employment. McCauley:Karyopharm Therapeutics: Employment, Equity Ownership. Carlson:Karyopharm Therapeutics: Employment. Savona:Karyopharm: Consultancy, Equity Ownership; Gilead: Consultancy; Incyte: Consultancy; Celgene: Consultancy. Kauffman:Karyopharm Therapeutics, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Shacham:Karyopharm Therapeutics, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Landesman:Karyopharm Therapeutics: Employment.

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Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.237.237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Michael P. Rettig ◽

Matthew Holt ◽

Julie Prior ◽

Sharon Shacham ◽

Michael Kauffman ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Employment Equity ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

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Calmodulin Inhibition Rescues the Effects of Ribosomal Protein Deficiency in in Vitro and In Vivo Diamond Blackfan Anemia Models

Blood ◽

10.1182/blood.v126.23.672.672 ◽

2015 ◽

Vol 126 (23) ◽

pp. 672-672

Author(s):

Elizabeth R Macari ◽

Alison Taylor ◽

David Raiser ◽

Kavitha Siva ◽

Katherine McGrath ◽

...

Keyword(s):

Ribosomal Protein ◽

Board Of Directors ◽

Transcriptional Activity ◽

Employment Equity ◽

Advisory Committees ◽

In Vivo Models ◽

Diamond Blackfan Anemia ◽

Equity Ownership

Abstract Ribosomal protein (RP) mutations are found in many diseases, including Diamond Blackfan anemia (DBA), where defective erythropoiesis, craniofacial abnormalities and increased cancer risk are major complications. RP mutations cause p53 activation through accumulation of free RPs that bind and sequester MDM2, the negative regulator of p53. We previously characterized a zebrafish mutant in rps29, a gene found mutated in DBA patients. Rps29-/- embryos have hematopoietic and endothelial defects, including decreased cmyb and flk1 expression and defects in hemoglobinization. Consistent with other animal models of RP dysfunction, p53 knockdown in rps29-/- embryos rescued these defects. To uncover novel compounds that correct the phenotypes of DBA, we performed a chemical screen in rps29-/- embryos. Several structurally distinct calmodulin (CaM) inhibitors successfully rescued hemoglobin (Hb) levels in the mutant embryo. To confirm that CaM inhibitors could rescue mammalian models of DBA, we applied them to human and murine models. Treating cord blood-derived CD34+ cells deficient in RPS19 with the CaM inhibitor, trifluoperazine (TFP), relieved the erythroid differentiation block. Injection of TFP in a DBA murine model significantly increased red blood cell number and Hb levels. Mechanistic studies in A549 cells infected with lentivirus expressing RPS19 shRNA demonstrated that TFP blocks p53 nuclear accumulation and induction of multiple p53 transcriptional target genes (p<0.05). Through p53 genetic manipulation, we determined that TFP inhibits p53 transcriptional activity through its c-terminal domain (CTD). Since this region has many residues that can be phosphorylated by CaM-dependent kinases, we hypothesized that TFP blocked phosphorylation of residues in the CTD. To test this hypothesis, phosphomimetic mutants were transfected into Saos2 cells and p53 transcriptional activity in response to TFP was evaluated using p21mRNA levels. TFP treatment of cells containing WT p53 or a transactivation domain mutant, S15D, resulted in a 4-fold reduction in p21 mRNA levels, while all four phosphomimetic mutants in the CTD had attenuated responses to TFP (<2-fold). The common CaM-dependent kinases that phosphorylate these CTD residues are Chk1 and Chk2. Investigation of the role of Chk1 and Chk2 found that a chk2 morpholino and multiple inhibitors of Chk2, but not Chk1, rescued Hb levels in the rps29-/- embryo (p<0.05). Chk2 inhibitors also mimic CaM inhibition in our in vitro assays. In conclusion, we have shown a novel mechanism by which CaM inhibitors mediate p53 activity through the CTD and can rescue the phenotypes of multiple in vitro and in vivo models of DBA. Our data strongly suggests that CaM or Chk2 inhibitors may be effective therapies for DBA patients, and a clinical trial is being planned with TFP. Disclosures Ebert: Genoptix: Consultancy, Patents & Royalties; H3 Biomedicine: Consultancy; Celgene: Consultancy. Zon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Selinexor Demonstrates Marked Synergy with Dexamethasone (Sel-Dex) in Preclinical Models and in Patients with Heavily Pretreated Refractory Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v124.21.4773.4773 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4773-4773 ◽

Cited By ~ 4

Author(s):

Christine I. Chen ◽

Martin Gutierrez ◽

David S. Siegel ◽

Joshua R. Richter ◽

Nina Wagner-Johnston ◽

...

Keyword(s):

Board Of Directors ◽

Transcriptional Activation ◽

Nuclear Export ◽

Research Funding ◽

Phase 1 ◽

Advisory Committees ◽

Nuclear Retention ◽

Heavily Pretreated ◽

Equity Ownership

Abstract Introduction: The nuclear export protein exportin 1, (XPO1) is overexpressed in a wide variety of cancers including MM and often correlate with poor prognosis. Selinexor (KPT-330) is an oral Selective Inhibitor of Nuclear Export (SINE) XPO1 antagonist in Phase 1 and 2 clinical studies. Selinexor forces nuclear retention and reactivation of tumor suppressor proteins (TSPs) and reduction of many proto-oncogenes, including MDM2, MYC and Cyclin D. In addition, selinexor potently deactivates NF-κB, through forced nuclear retention of IκBα. Together these effects induce selective apoptosis in MM cells and inhibition of NF-κB dependent osteoclast activation. XPO1 is also responsible for nuclear export of the glucocorticoid receptor (GR). We hypothesized that selinexor will enhance the activity of dexamethasone (DEX)-bound GR, resulting in synergistic tumor cell killing. Methods: In vitro tumor cell viability measurements were based on MTT (CellTiter 96¨/Promega) and combination indices were calculated using CalcuSyn software. For xenograft studies, utilized NOD-SCID mice with subcutaneous inoculation of MM.1s cells. GR nuclear localization was measured with immunofluorescent anti-GR (phosphor-S211) antibody and quantitative imaging. To assess GR transcriptional activation, GR binding to a GCR consensus sequence was measured in nuclear extracts using an ELISA method (GR ELISA kit/Affymetrix). Patients (pts) with heavily pretreated refractory MM were dosed with oral selinexor at doses of up to 60 mg/m2 (8-10 doses/4 wk cycle) as part of a Phase 1 program in advanced hematological malignancies. Response we defined based on the IMWG criteria. The effect of combining DEX with selinexor was analyzed in all pts who received selinexor at moderate to high doses (30-60 mg/m2). Safety and efficacy were analyzed separately in three groups: no DEX, <20 mg DEX and 20 mgs DEX. Results: In MM.1s cells Sel-Dex showed synergy for nuclear retention of the DEX activated GR (Ser211-phosphorylated) and concomitant GR transcriptional activation. Sel-Dex showed highly synergistic cytotoxicity in MM.1s cells in vitro and in vivo, with a corresponding increase in apoptosis. Selinexor alone was potently cytotoxic in the DEX resistant MM cell lines MM.1R and ANBL6, but addition of DEX provided no additional effect. Twenty-eight pts with heavily pretreated refractory MM (16 M, 12 F; median age 62; ECOG PS 0/1: 7/21; median prior regimens: 6) received selinexor at 30 – 60 mg/m2 with either 0, <20, or 20 mgs DEX. All pts have received a proteasome inhibitor and an Imid and the majority of the pts have received pomalidomide (68%) and/or carfilzomib (36%). The most common Grade 1/2 AEs for these three groups were: nausea (82%/86%/70%), fatigue (55%/86%/40%), anorexia (36%/71%/60%), and vomiting (36%/57%/10%). Of the 28 pts treated; 10 heavily pretreated refractory MM pts treated with a combination of selinexor (45 mg/m2 twice weekly) and DEX (20 mg with each selinexor dose) were found to have dramatically improved disease response (n=10, ORR 60%), with one stringent complete response (sCR, 10%), 5 partial responses (PR, 50%) and clinical benefit rate (CBR) rate of 80% (Figure 1). Treatment with ³30mg/m2 selinexor and <20 mg DEX (n=7), resulted in ORR of 14% and CBR of 86%, while treatment with selinexor (30-60 mg/m2) without DEX (n=12) showed best response of stable disease (50%). Sel-Dex was also associated with an increase in time on study relative to selinexor alone, with 7 of out 10 pts in the 20 mg DEX combo group still on study (11-25 weeks). Five additional pts were treated with selinexor at a dose of 60 mg/m2 in combination with 20 mg DEX. Response evaluation is pending. Conclusions: Sel-Dex combination is markedly synergistic in preclinical models, which is supported by the preliminary clinical data presented. One potential mechanism underlying this synergy is the amplification of GR activity due the combined effects of selinexor-induced nuclear retention of activated GR coupled with DEX-mediated GR agonism. These results provide a promising basis for the continuing study of Sel-Dex for treatment of pts with refractory MM. Phase 2 studies of Sel-Dex in pts with MM refractory to both pomalidomide and carfilzomib are planned for early 2015. Disclosures Chen: Celgene: Honoraria; Janssen: Honoraria. Off Label Use: Lenalidomide maintenance therapy after ASCT. Gutierrez:Senesco: PI Other. Siegel:Celgene, Millennium, Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baz:Celgene: Research Funding; Millennium: Research Funding; Bristol Myers Squibb: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding. Kukreti:Celgene: Honoraria. Azmi:Karyopharm Therpeutics: Research Funding. Kashyap:Karyopharm Therapeutics: Employment. Landesman:Karyopharm Therapeutics: Employment. Marshall:Karyopharm Therpeutics: Employment. McCartney:Karyopharm Therpeutics: Employment. Saint-Martin:Karyopharm Therpeutics: Employment. Norori:Karyopharm Therpeutics: Consultancy. Savona:Karyopharm Therpeutics: Membership on an entity's Board of Directors or advisory committees. Rashal:Karyopharm Therapeutics: Employment. Carlson:Karyopharm Therapeutics: Employment. Mirza:Karyopharm Therpeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics: Employment, Equity Ownership. Reece:Millennium: Honoraria, Research Funding; Millennium: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Merck: Research Funding; Merck: Research Funding; BMS: Research Funding; BMS: Research Funding; Novartis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen : Honoraria; Amgen : Honoraria.

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Effects Of Inhibition Of XPO1/CRM1-Dependent Nuclear Export By Selinexor (KPT-330), Alone and In Combination With Carfilzomib (CFZ), On Apoptosis and Autophagy In Multiple Myeloma (MM)

Blood ◽

10.1182/blood.v122.21.279.279 ◽

2013 ◽

Vol 122 (21) ◽

pp. 279-279 ◽

Cited By ~ 1

Author(s):

Shaun Rosebeck ◽

Malathi Kandarpa ◽

Mattina M. Alonge ◽

Jagoda Jasielec ◽

Dominik Dytfeld ◽

...

Keyword(s):

Cell Death ◽

Cancer Cells ◽

Board Of Directors ◽

Nuclear Export ◽

Research Funding ◽

Proteasome Inhibition ◽

Employment Equity ◽

Advisory Committees ◽

Nuclear Retention ◽

Equity Ownership

Abstract Introduction Exportin 1 (XPO1/CRM1) is the sole transporter of most tumor suppressor proteins (TSP) from the nucleus to the cytoplasm. Small molecule selective inhibitors of nuclear export (SINE) block XPO1-mediated nuclear export, leading to nuclear retention of TSP, inducing cancer cell death and sensitizing cancer cells to other cytotoxic drugs. Although the cytotoxic and apoptotic effects of SINE on different cancer cells have now been established, the mechanism of cell death is still not fully understood. Recently, autophagy emerged as a possible cell death pathway and alternate to the ubiquitin-proteasome pathway (UPP) by which excess and/or dysfunctional proteins and organelles are degraded and recycled. MM cells require basal autophagy for survival and caspase 10 protease activity is required to limit autophagic cell death. The possibility that autophagy may be involved in the mechanism of action of SINE is supported by observations that knockdown of XPO1 can promote autophagy and that cytoplasmic p53 can repress autophagy. In this study, we evaluated the contribution of autophagy to the effects of Selinexor (KPT-330), a SINE currently in two phase I clinical trials, on MM cell cytotoxicity. Because proteasome inhibition can also induce autophagy, we hypothesized that the combination of Selinexor and CFZ, a next generation irreversible proteasome inhibitor approved for treatment of MM, may synergistically augment cytotoxicity in MM cells. Methods Plasma cells (PC) were purified from consented MM patient bone marrow aspirates using EasySep (Stem Cell Technology). PC purity (>80%) was determined by Wright-Giemsa staining of cytospins. Human myeloma cell lines (HMCL) NCI-H929, RPMI-8226, MM1.S and MM1.R were cultured in RPMI1640 with 10% FBS. IC50 values were determined using GraphPad Prism. Drug combination efficacy was determined using CalcuSyn (Biosoft). Combination index (CI) values <1.0 indicate synergy. Transcription factor profiling plate array II was from Signosis (Sunnyvale, CA). Otherwise, our studies used standard cellular and molecular biology techniques. Results Selinexor caused significant cytotoxicity in HMCL (IC50 10-100 nM), induced cell cycle arrest in G1, and promoted apoptotic cell death typified by caspase activation, DNA fragmentation, and Annexin V binding. Importantly, purified PC from newly-diagnosed MM patients were also sensitive to the cytotoxic effects of Selinexor. HMCL treated with KPT-330 also exhibited nuclear retention of p53. To gain insight into more global effects of SINE treatment, we assayed DNA binding activity of approx. 100 different transcription factors. TSP, such as Rb, p53, and EGR-1, exhibited increased activity, whereas proto-oncogenes, including NF-κB, Myc, and Myb, were inactivated in response to KPT-330. Western blot analysis of inactivated targets showed nearly total loss of protein. Unlike XPO1, which is degraded via the UPP upon treatment with SINE, loss of Myc and NF-κB subunits, including RelA, RelB, and p52, could not be reversed by proteasome inhibition. Instead, we found that Selinexor treatment induced markers of autophagy, including LC3B induction and processing, and promoted loss of caspase 10, which is associated with autophagic cell death in MM. Importantly, we have determined that the combination of Selinexor and CFZ results in synergistic cell death (CI 0.2-0.6) characterized by enhanced induction of both apoptosis and autophagy in HMCL. The effects of Selinexor and CFZ were also evaluated in NOD-SCID mice inoculated subcutaneously with NCI-H929 cells. Mice were treated 3 times weekly per oswith Selinexor (5 or 10 mg/kg) alone and in combination with twice-weekly IV-administered CFZ (1.5 or 3 mg/kg). After 16 days of treatment, high doses of either CFZ or Selinexor alone moderately inhibit tumor growth. Treatment with the combination of CFZ and Selinexor at all dose levels was more effective than single agent treatment, and high dose combination treatment completely impaired xenograft tumor growth with good tolerability. Conclusion Our studies are the first to suggest that Selinexor-induced cell death correlates with both apoptosis and autophagy, and that both cell death pathways are enhanced in response to combined treatment with CFZ. Overall, our pre-clinical study provides strong rationale for evaluation of Selinexor in combination with CFZ for the treatment of MM. Disclosures: McCauley: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Jakubowiak:BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Combination Treatment with 5F9 and Azacitidine Enhances Phagocytic Elimination of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-120170 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2729-2729 ◽

Cited By ~ 8

Author(s):

Dongdong Feng ◽

Phung Gip ◽

Kelly Marie McKenna ◽

Feifei Zhao ◽

Ofelia Mata ◽

...

Keyword(s):

Cancer Cells ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Bioluminescence Imaging ◽

Employment Equity ◽

Advisory Committees ◽

Human Macrophages ◽

Equity Ownership

Abstract CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential. In this study, we hypothesized that combining 5F9 with azacytidine (AZA) would enhance therapeutic efficacy against AML. AZA (Vidaza®) is a hypomethylating and chemotherapeutic agent indicated for AML. AZA's anti-cancer mechanism of action is believed to be twofold, the first being induction of DNA demethylation and the second being its anti-metabolite activity. Interestingly, it has also been found that AZA can increase the expression of the anti-phagocytic signal, CD47, and the pro-phagocytic signal, calreticulin, in myeloid malignancies. Based on these previous findings, we hypothesized that AML cells may be more efficaciously eliminated using a combination of AZA and 5F9 through enhancement of AML cell phagocytosis. We first tested this hypothesis using an in vitro phagocytosis assay. AML cells (i.e. GFP-expressing HL60 cells) were incubated for 24 hours with 3µM AZA and afterwards, the HL60-GFP cells were co-cultured for 2 hours with either human macrophages plus IgG4 control or 5F9 (10µg/ml). Phagocytosis of HL60 AML cells was calculated as a percentage of GFP-positive macrophages (i.e. the amount of macrophages that engulfed GFP-positive HL60 cells), compared to total number of macrophages. Results were normalized to a condition that produced the maximum amount of phagocytosis (100%). We found that the combination of AZA with 5F9 enhanced the human macrophage-mediated phagocytic elimination of HL60-GFP cells compared to either agent alone (Fig. 1). Next, we asked whether we could confirm our in vitro findings in vivo utilizing an aggressive AML xenograft mouse model. HL60-GFP cells (500,000 cells/per mouse) expressing luciferase were engrafted by intravenous injection into 6 - 8 week old immune-deficient NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. Three days post engraftment (PE), bioluminescence imaging was performed to assess AML engraftment based on total flux (photons/sec). Animals were randomized based on these values into 6 treatment cohorts with 8 animals per group. Treatment was performed as follows: (1) control (PBS) was initiated on day 4 PE and continued for 14 consecutive daily doses; (2) AZA (7.5 mg/kg) was initiated on day 4 PE and continued for 5 consecutive daily doses; (3) two cohorts of 5F9 (10mg/kg) were initiated at day 4 or day 7 PE and continued for 14 consecutive daily doses; and (4) two combination cohorts of AZA with 5F9 were initiated according to the 5F9 monotherapy dosing regimens. Routine bioluminescence imaging was performed during treatment and for several months after to assess AML burden and reoccurrence. Both combination cohorts inhibited AML growth as early as day 10 PE, and maintained elimination of growth and overall survival up to 255 days PE. In contrast, the AZA and 5F9 monotherapies initiated at day 7 PE (D7), decreased AML growth at day 10 PE, but failed to produce a durable response. Notably, as the AML expanded, all animals from the AZA cohort died by 46 days PE, and all animals from the 5F9 cohort died by 61 days PE. Of the 8 animals from the 5F9 cohort that received treatment on day 4 PE, only two animals demonstrated progressive disease and did not survive. The remaining animals from this cohort had no detectable AML cancer cells (Fig 2). In summary, the combination of 5F9 with AZA significantly enhanced the phagocytic elimination of AML cells by human macrophages in vitro, enhanced clearance of AML in vivo, and prolonged survival compared to single agent treatment with AZA or 5F9. These results support the rationale for investigating a combinatorial treatment of 5F9 and AZA in patients with AML. A clinical trial with this combination in patients with AML is currently ongoing (NCT03248479). Disclosures Feng: Forty Seven Inc: Employment, Equity Ownership. Gip:Forty Seven Inc: Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Zhao:Forty Seven Inc: Consultancy. Mata:Forty Seven Inc: Employment, Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc: Consultancy, Equity Ownership. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

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ACY-241, a Novel, HDAC6 Selective Inhibitor: Synergy with Immunomodulatory (IMiD®) Drugs in Multiple Myeloma (MM) Cells and Early Clinical Results (ACE-MM-200 Study)

Blood ◽

10.1182/blood.v126.23.3040.3040 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3040-3040 ◽

Cited By ~ 8

Author(s):

Ruben Niesvizky ◽

Paul G. Richardson ◽

Nashat Y. Gabrail ◽

Sumit Madan ◽

Andrew J. Yee ◽

...

Keyword(s):

Clinical Trial ◽

Combination Therapy ◽

Board Of Directors ◽

Research Funding ◽

Selective Inhibitor ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Histone deacetylase (HDAC) enzymes are attractive therapeutic targets in oncology, but non-selective HDAC inhibitors have led to dose-limiting toxicities in patients, particularly in combination with other therapeutic agents. Ricolinostat (ACY-1215), a first-in-class orally available HDAC inhibitor that is 11-fold selective for HDAC6, synergizes in vitro and in vivo in models of MM and lymphoma with bortezomib (Santo, Blood, 2012; Amengual, Clin Cancer Res, 2015) or carfilzomib (Mishima, Br J Haematol, 2015; Dasmahapatra, Mol Cancer Ther, 2014). Furthermore, ricolinostat has demonstrated an excellent safety and tolerability profile in phase I trials as an oral liquid formulation (Raje, Haematologica, 2014, Suppl 1). We have now identified ACY-241 as a structurally related and orally available selective inhibitor of HDAC6 that is undergoing clinical evaluation in tablet form. In combination with ricolinostat, the immunomodulatory (IMiD®) class of drugs, including lenalidomide (Len) and pomalidomide (Pom), exhibit striking anti-myeloma properties in a variety of MM models (Quayle, AACR, 2014) and have demonstrated clinical activity in MM patients (Yee, ASH, 2014). In support of our ongoing development of ACY-241, we show here that combination with either Len or Pom leads to synergistic decrease in MM cell viability in vitro. Time course studies demonstrated cell cycle arrest followed by progressive induction of apoptosis after prolonged exposure to Len or Pom. Notably, the addition of ACY-241 to either Len or Pom resulted in synergistic increases in apoptosis of MM cells. At the molecular level, treatment with IMiDs reduced expression of the critical transcription factors MYC and IRF4, which was further reduced by combination treatment with ACY-241. Current studies are exploring the molecular mechanism underlying this effect, which may be a consequence of low level inhibition of HDAC1, 2, and 3 by ACY-241. Prolonged treatment with ACY-241 plus Pom was well tolerated in vivo with no evidence of toxicity, and the combination resulted in a significant extension of survival in a xenograft model of MM. Given the comparable tolerability profiles of ricolinostat and ACY-241 and the similar preclinical activity in combination with IMiDs, a clinical trial (NCT02400242) is currently evaluating ACY-241 in combination with Pom and low-dose dexamethasone in MM patients. Predicated upon the clinical experience with ricolinostat and the non-clinical pharmacokinetics of ACY-241, we designed an expedited first-in-human phase 1a/1b clinical trial of a single cycle of ACY-241 monotherapy followed by ACY-241 in combination with Pom and dexamethasone in MM patients. A merged monotherapy/combination trial design was chosen to grant patients access to combination therapy with an established regimen while enabling insight into the safety, pharmacokinetics, and pharmacodynamics of ACY-241 monotherapy. Patients with relapsed or relapsed-and-refractory MM previously treated with at least two cycles of Len and a proteasome inhibitor were eligible for this trial. The first patient was enrolled in June 2015. This patient tolerated monotherapy well and pharmacokinetics showed maximal plasma levels of ACY-241 in the micromolar range, consistent with predictions. An update on enrollment, pharmacokinetic and pharmacodynamic profiles as well as safety of monotherapy and combination therapy will be provided. Disclosures Niesvizky: Celgene: Consultancy, Speakers Bureau. Richardson:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Gabrail:Onyx: Honoraria, Speakers Bureau; BI: Honoraria, Speakers Bureau; Janssen: Speakers Bureau; Sanofi: Honoraria, Speakers Bureau. Madan:Onyx: Speakers Bureau; Celgene: Speakers Bureau. Quayle:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Almeciga-Pinto:Acetylon Pharmaceuticals, Inc: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Houston:Acetylon Pharmaceuticals, Inc: Employment. Hayes:Acetylon Pharmaceuticals, Inc: Employment. Van Duzer:Acetylon Pharmaceuticals, Inc: Employment. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Trede:Acetylon Pharmaceuticals, Inc: Employment. Raje:Acetylon: Research Funding; Celgene Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy; Millenium: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; Onyx: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy.

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Anti-KIR Antibody Enhancement Of Anti-Lymphoma Activity Of Natural Killer Cells As Monotherapy and In Combination With Anti-CD20 Antibodies

Blood ◽

10.1182/blood.v122.21.4417.4417 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4417-4417 ◽

Cited By ~ 1

Author(s):

Holbrook E Kohrt ◽

Ariane Thielens ◽

Aurelien Marabelle ◽

Idit Sagiv Barfi ◽

Caroline Sola ◽

...

Keyword(s):

Board Of Directors ◽

Nk Cell ◽

Dependent Manner ◽

Employment Equity ◽

Advisory Committees ◽

Potential Efficacy ◽

Equity Ownership ◽

Anti Cd20

Natural killer (NK) cells mediate anti-lymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of NK cell's efficacy by spontaneous cytoxicity. Using a killer cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with MHC class I antigens on lymphoma by anti-KIR antibodies prevents a tolerogenic interaction and augments NK cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in syngeneic and KIR transgenic murine lymphoma models. Specifically targeting murine NK cells in vitro, anti-Ly49C/I F(ab')2 increased anti-CD20 mAb-mediated NK cell degranulation as measured by CD107a mobilization and interferon-γ release, as well as increased cytotoxicity as assessed by chromium release. In the syngeneic EL4-huCD20 lymphoma model, anti-Ly49C/I F(ab')2 enhanced the anti-lymphoma activity of anti-CD20 mAb in vivo (Fig 1A-1B) and was NK cell-dependent with efficacy abrogated by NK cell depletion with anti-Asialo-GM1. To validate these observations and the potential efficacy of a fully human anti-KIR mAb (IPH2101, lirilumab), we demonstrated, in vitro, dose-dependent KIR2DL3 saturation and tumor lysis following blockade of KIR2DL3/HLA-C with lirilumab. In the transgenic KIR murine model, lirilumab therapy improved survival in an NK cell-dependent manner in both a prophylactic and therapeutic HLA+ (221 HLA-Cw3) lymphoma model. In combination, lirilumab therapy synergistically enhanced rituximab's anti-lymphoma efficacy in vivo in an NK cell-dependent manner (Fig 2A-C). These results support a therapeutic strategy of combination, rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor targeting therapy with an NK cell agonist thus stimulating the post-rituximab anti-lymphoma immune response. Disclosures: Thielens: Innate Pharma: Employment, Equity Ownership. Sola:Innate Pharma: Employment, Equity Ownership. Chanuc:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment. Bonnafous:Innate Pharma: Employment, Equity Ownership. Vivier:Innate Pharma: Membership on an entity’s Board of Directors or advisory committees. Romagne:Innate Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Andre:Innate-Pharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership.

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SEL120 - a First-in-Class CDK8/19 Inhibitor As a Novel Option for the Treatment of Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndrome - Data from Preclinical Studies and Introduction to a Phase Ib Clinical Trial

Blood ◽

10.1182/blood-2019-127835 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2651-2651

Author(s):

Gautam M. Borthakur ◽

William B. Donnellan ◽

Scott R. Solomon ◽

Camille Abboud ◽

Aziz Nazha ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Preclinical Studies ◽

Employment Equity ◽

Advisory Committees ◽

Phase Ib ◽

Equity Ownership

Deregulated transcription, one of the key features of acute myelogenous leukemia (AML), remains largely unactionable by recently approved therapies. Preclinical studies indicate that targeting Cyclin Dependent Kinase 8 (CDK8) may be a novel therapeutic strategy for AML. CDK8 and its paralog CDK19, restrain activation of super-enhancer-associated tumor suppressors and lineage commitment genes in AML cells. SEL120 is a first-in-class, specific and selective inhibitor of CDK8/CDK19 and has shown activity in preclinical AML models. Preclinical characterization of SEL120 demonstrated a unique mechanism of action, related to known functions of CDK8 in the regulation of transcription. Cells sensitive to SEL120 show enrichment for leukemia stem cells (LSCs) signatures and higher signal transducer and activator of transcription 5 (STAT5) levels. Treatment with SEL120 reduced STAT5 phosphorylation in sensitive cell lines both in vitro and in vivo. Transcriptomic analysis of AML cells revealed that SEL120 regulated genes involved in lineage controlling functions. Specifically, trimethylation of lysine 27 on histone H3 (H3K27me3) by the polycomb repressive complex 2 (PRC2), was proposed to maintain the stemness of LSCs by preventing differentiation. Derepression of lineage commitment genes marked with H3K27me3, was one of the earliest transcriptional events in sensitive cells treated with SEL120. Consistent with its transcriptional effects, SEL120 induced differentiation of AML cells. Additionally SEL120 significantly repressed the MYC Proto-Oncogene-dependent transcriptomic signatures. Efficacy of SEL120 in AML was confirmed in models with high translational potential, including patient-derived AML cells (PDC) in vitro and in vivo. PDCs treated with SEL120 showed reduced viability, induction of apoptotic cell death and differentiation commitment. Administration of SEL120 in orthotopic AML patient-derived xenograft models reduced tumor burden to the level undetectable by flow cytometry, decreased splenomegaly and resulted in partial bone marrow recovery. CLI120-001 is a first-in-human, open-label, multi-center, modified 3+3 dose escalation phase Ib study of SEL120 with a dose-escalation cohort (DC) followed by an enrichment cohort (EC) in adult patients with AML or high-risk myelodysplastic syndrome who have relapsed or refractory disease and have received no more than 3 prior lines of therapy. Other key inclusion criteria are Eastern Cooperative Oncology Group performance status of 0-2, white blood cell (WBC) count <10000/µL (prior hydroxyurea is permitted to reduce WBC), platelet count >10000/µL, adequate organ function defined as: aspartate aminotransferase and alanine aminotransferase ≤3x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine clearance ≥60 ml/min, left ventricular ejection fraction ≥40%. Major exclusion criteria include Q to T wave interval corrected for heart rate (QTc) ≥450 ms, taking concomitant medications that are known to be strong inhibitors or inducers of cytochrome P450 1A2 or that can prolong QTc and/or cause torsade de pointes. The primary objective of the study is to assess safety and tolerability of SEL120 and to establish the recommended dose (RD) for further clinical development. Secondary objectives include evaluation of preliminary anti-leukemic activity and characterization of the pharmacokinetic profile of SEL120. The exploratory objective is to assess pharmacodynamics of SEL120 by using relevant biomarkers, including STAT5 pS726, transcriptional profiling by RNAseq and immunophenotypic changes related to stemness and differentiation of AML cells. SEL120 is administered as a single oral dose every other day for a total of 7 doses i.e. on days 1, 3, 5, 7, 9, 11 and 13, in a 21-day treatment cycle. Patients receive SEL120 until disease progression, unacceptable toxicity, or withdrawal of consent. The DC is now enrolling patients who are treated at dose levels defined by a modified Fibonacci sequence, and will end with selection of the RD based on all available study data. In the EC, additional patients will be treated at the RD to further support the evaluation of the RD of SEL120 monotherapy. The study is currently running in the United States and is planned to be completed in 2020. The ClinicalTrials.gov Identifier: NCT04021368. Disclosures Borthakur: Eli Lilly and Co.: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agensys: Research Funding; Eisai: Research Funding; Oncoceutics: Research Funding; Oncoceutics, Inc.: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; NKarta: Consultancy; Cyclacel: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Bayer Healthcare AG: Research Funding; Novartis: Research Funding; Cantargia AB: Research Funding; Strategia Therapeutics: Research Funding; BMS: Research Funding; PTC Therapeutics: Consultancy; Xbiotech USA: Research Funding; AbbVie: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Merck: Research Funding; AstraZeneca: Research Funding; Tetralogic Pharmaceuticals: Research Funding. Abboud:Jazz Pharma: Speakers Bureau; Novartis: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017), Research Funding; Agios: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017); Tetraphase Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; NKarta: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. Nazha:Abbvie: Consultancy; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Bialas:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Chrom:Selvita S.A.: Employment. Rzymski:Selvita S.A.: Employment, Equity Ownership. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Blockade of XBP1 Splicing by Inhibition of IRE1α Is a Promising Therapeutic Option in Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.133.133 ◽

2011 ◽

Vol 118 (21) ◽

pp. 133-133 ◽

Cited By ~ 2

Author(s):

Naoya Mimura ◽

Mariateresa Fulciniti ◽

Gullu Gorgun ◽

Yu-Tzu Tai ◽

Diana D. Cirstea ◽

...

Keyword(s):

Growth Inhibition ◽

Er Stress ◽

Board Of Directors ◽

Tumor Cells ◽

Therapeutic Option ◽

Rt Pcr ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership

Abstract Abstract 133 Multiple myeloma (MM) cells are characterized by high protein synthesis resulting in chronic endoplasmic reticulum (ER) stress, which is adaptively managed by the unfolded protein response (UPR). Therefore blockade of UPR could provide a novel therapeutic option in MM. Upon UPR, inositol-requiring enzyme 1α (IRE1α) is activated by auto-phosphorylation, resulting in activation of its endoribonuclease domain to cleave XBP1 mRNA from XBP1 unspliced form (XBP1u: inactive) to generate the XBP1 spliced form (XBP1s: active). XBP1s protein in turn regulates genes responsible for protein folding and degradation, playing a pro-survival signaling role in the UPR. In this study, we specifically examined whether IRE1α-XBP1 pathway is a potential therapeutic target in MM. We first examined the biologic significance of IRE1α by knockdown using lentiviral shRNA and observed significant growth inhibition in IRE1α knockdown cells. We next examined the impact of inhibition of XBP1 splicing using a novel small molecule IRE1α endoribonuclease domain inhibitor MKC-3946 (MannKind, Valencia CA). MKC-3946 blocked not only the basal level, but also inducible (by tunicamycin) XBP1s, evidenced by RT-PCR analysis in RPMI8226 cells, without affecting phosphorylation of IRE1α. Importantly, MKC-3946 also inhibited XBP1s in primary tumor cells from MM patients. We also confirmed functional inhibition of XBP1s, with target genes including SEC61A1, p58IPK, and ERdj4 downregulated by MKC-3946 treatment. Importantly, MKC-3946 triggered growth inhibition in MM cell lines, without toxicity in normal mononuclear cells. Furthermore, it significantly enhanced cytotoxicity induced by bortezomib or 17-AAG in RPMI8226 and INA6 cells, as well as primary tumor cells from MM patients. Both bortezomib and 17-AAG induced ER stress with XBP1s, which was markedly blocked by MKC-3946. Moreover, apoptosis induced by bortezomib or 17-AAG was enhanced by MKC-3946, associated with increased CHOP mRNA and protein, a proapoptotic factor triggered by ER stress. We next demonstrated that XBP1s was induced by bortezomib in INA6 cells co-cultured with bone marrow (BM) stromal cells, which was inhibited by MKC-3946, associated with enhanced cytotoxicity induced by the combination. Finally, MKC-3946 inhibited XBP1s in a model of in vivo ER stress induced by tunicamycin. To evaluate the anti-MM effect of MKC-3946, we used the subcutaneous RPMI8226 xenograft model in mice. MKC-3946 significantly reduced MM tumor growth in the treatment versus control group, associated with prolonged overall survival. We also confirmed that MKC-3946 treatment significantly inhibited XBP1s in excised tumors, assessed by RT-PCR. In order to examine the activity of MKC-3946 on MM cell growth in the context of the human BM microenvironment in vivo, we used the SCID-hu model, in which INA6 cells are directly injected into a human bone chip implanted subcutaneously in SCID-mice. MKC-3946 treatment significantly inhibited tumor growth compared with vehicle control. Moreover, XBP1s in excised tumor cells was inhibited, evidenced by RT-PCR. In conclusion, these data demonstrate that blockade of XBP1s by MKC-3946 triggers MM cell growth inhibition in vivo and prolongs host survival. Taken together, our results demonstrate that blockade of XBP1 splicing by inhibition of IRE1α endoribonuclease domain is a potential novel therapeutic option in MM. Disclosures: Tam: MannKind Corporation: Employment, Equity Ownership. Zeng:MannKind Corporation: Employment, Equity Ownership. Patterson:MannKind Corporation: Employment, Equity Ownership. Richardson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Munshi:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; MannKind: Membership on an entity's Board of Directors or advisory committees.

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