Combination Therapy with Bortezomib or Carfilzomib and Selinexor Induces Nuclear Localization of Ikbα and Overcomes Acquired Proteasome Inhibitor Resistance in Human Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3299-3299
Author(s):  
Joel G Turner ◽  
Trinayan Kashyap ◽  
Jana L Dawson ◽  
Juan A Gomez ◽  
Alexis Bauer ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Transcriptional Activity ◽  
Immunofluorescence Microscopy ◽  
Single Agent ◽  
Advisory Committees ◽  
Proximity Ligation ◽  
Nsg Mice ◽  
Nfkb Pathway

Abstract Introduction: Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired-resistance in MM. Materials and Methods: PI-resistant myeloma cell lines, RPMI8226-B25 and U226 PSR, and their respective parental cell lines RPMI8226 and U266, were treated both in vitro with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. In vivo studies using U266 and U266PSR tumors were performed in NOD/SCID-gamma (NSG) mice. Mice were treated with selinexor/bortezomib and single agents. Bone marrow biopsies from refractory myeloma patients were treated ex vivo with selinexor/bortezomib or selinexor/carfilzomib and assayed for apoptosis. Mechanistic studies included NFkB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry and proximity-ligation assay. IkBα knockdown and NFkB transcriptional activity were measured in selinexor/bortezomib treated MM cells. Results: We found that selinexor restored sensitivity of PI-resistant RPMI8226-B25 and U266PSR MM cells to bortezomib (P = 0.00055) and carfilzomib (P = 0.0017). Bortezomib, when combined with selinexor reduced U266 MM tumor growth versus single-agent bortezomib (P = 0.022) in NSG mice. NSG mice challenged with PI-resistant U266PSR MM tumors also had reduced tumor growth with selinexor/bortezomib as compared to single agent bortezomib (P = 0.0006). Combining bortezomib and selinexor improved survival in mice with U266 MM tumors (P = 0.0072) and PI-resistant U266PSR when compared to single-agent bortezomib (P = 0.0072). Myeloma cells from PI-refractory MM patients (n=14) were sensitized by selinexor to bortezomib (P = 0.002) and carfilzomib (P = 0.001) without affecting non-myeloma cells. Immunofluorescence microscopy of PI-resistant human MM cell lines found a greater than 212% increase in IkBα when compared to untreated cells (confirmed by Western blot). A similar increase in IkBα immunofluorescence was found in newly diagnosed, relapsed and refractory patient MM cells. ImageStream analyses of MM cells showed an increase in total and nuclear IkBα from selinexor/bortezomib exposure. Proximity-ligation assays showed that IkBα-NFkB-complexes were increased 12-fold in bortezomib/selinexor treated MM cells. IkBα knockdown abrogated selinexor/bortezomib induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFkB transcriptional activity in addition to a reduction of NFkB induced IAP-1, IAP-2, BCL-2, cyclin D2 and c-myc protein expression.. Conclusions: Selinexor, when used with bortezomib or carfilzomib has the potential to overcome proteasome-inhibitor drug-resistance in MM. Sensitization may be due to inactivation of the NFkB pathway by IkBα. Selinexor, an orally active selective inhibitor of XPO1-mediated nuclear export (SINE), is currently undergoing phase I/II studies in a variety of indications, including a combination with carfilzomib, in both relapsed and refractory MM patients (NCT02199665). The results presented in this study support combinatorial clinical trials in relapsed and refractory MM that utilize PI therapies. Disclosures Kashyap: Karyopharm Therapeutics: Employment, Equity Ownership. Shain:Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Novartis: Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Landesman:Karyopharm Therapeutics Inc: Employment, Other: stockholder.

Novel Inhibitors Of CRM1/XPO1 Nuclear Exporter Exhibit Striking Activity Against AML “primagrafts,” Including AML Leukemia Initiating Cells, While Sparing Normal Hematopoietic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3932-3932
Author(s):  
Julia Etchin ◽  
Bonnie Thi Le ◽  
Alex Kentsis ◽  
Richard M. Stone ◽  
Dilara McCauley ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Hematopoietic Cells ◽  
Complex Karyotype ◽  
Employment Equity ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Equity Ownership

Abstract Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are also toxic to normal tissues, prompting the need to develop new targeted therapies. One attractive cellular pathway with therapeutic potential is nuclear export, which is mediated in part by nuclear exporter CRM1/XPO1. XPO1 mediates the transport of ∼220 proteins and several mRNAs and is the sole nuclear exporter of the major tumor suppressor and growth regulatory proteins p53, p73, FOXO, IkB/NF-kB, Rb, p21, and NPM. Our findings demonstrate that novel irreversible inhibitors of XPO1, termed Selective Inhibitors of Nuclear Export, or SINE, induce rapid apoptosis in 12 AML and 14 T-ALL cell lines with IC50s of 15-474 nM. In the SINE-sensitive cell lines, BCL2 overexpression suppresses SINE-induced apoptosis, indicating its intrinsic pathway mediation. Oral administration of clinical XPO1 inhibitor, Selinexor (KPT-330), at 15 or 25 mg/kg, induced remarkable growth suppression in MV4-11 human AML cells and MOLT-4 human T-ALL cells engrafted in immunodeficient NSG mice with negligible toxicity to normal mouse hematopoietic cells after 35 days of treatment. Bone marrow biopsies of selinexor - treated mice were remarkable in that they showed normal hematopoietic cell morphology and cellularity after 35 days of treatment. Significant survival benefit was observed in mice treated with selinexor, compared to vehicle-treated mice. Selinexor is now in Phase 1 clinical trial in patients with AML and other hematological malignancies (NCT01607892). Recently, we have established primagraft models of AML, using primary leukemia blasts isolated from AML patients at diagnosis transplanted into immunocompromised NSG mice. We demonstrated that selinexor exhibits striking anti-leukemic activity against different subtypes of primary AML, including AML-M4; FLT3-ITD and complex karyotype subtypes of the disease. To determine whether selinexor targets leukemia-initiating cells (LICs) of primary AML, we re-transplanted serial dilutions of human CD45+ cells isolated from leukemic mice treated with either vehicle or selinexor. The preliminary results of our re-population assays indicate that selinexor greatly diminished LIC frequency in AML-M4; FLT3-ITD AML (∼6 fold) and complex karyotype disease (∼100 fold). These findings demonstrate that selinexor may represent a novel targeted therapy for the treatment of AML, which spares normal hematopoietic stem and progenitor cells. Disclosures: McCauley: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. 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.

The Combination Of Hypomethylating Agents and Histone Deacetylase Inhibitors(HDACi) Are Synergistically Cytotoxic and Reverse The Malignant Phenotype In Preclinical Models Of T-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 646-646 ◽  
Author(s):  
Owen A. O'Connor ◽  
Enrica Marchi ◽  
Kelly Zullo ◽  
Luigi Scotto ◽  
Jennifer E. Amengual ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Single Agent ◽  
Hematologic Malignancies ◽  
Advisory Committees ◽  
Methylation Array ◽  
Simultaneous Exposure ◽  
The Mean

Abstract Both HDAC inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) are known to influence global expression patterns in hematologic malignancies. Little is known about the combination of these two drug classes in lymphoid malignancies. HDACIs have marked single agent activity in the T- cell lymphomas (TCL), although the mechanism of action is not well defined. DNMTIs affect cytosine methylation of genomic DNA and have activity mainly restricted to the myeloid derived hematologic malignancies. The single agent efficacy and synergistic interaction of a panel of HDACIs (panobinostat, belinostat, romidepsin and vorinostat) and DNMTIs (decitabine (DEC), 5-azacytadine (5-AZA)) was evaluated in models of TCL. The molecular basis for the synergistic effect of HDACIs and DNMTIs was evaluated by gene expression profiling (GEP) and CpG methylation CTCL. Single agent concentration and time effect relationships were generated for 2 CTCL (HH, H9) and 2 T-ALL (P12, PF382) cell lines. Romidepsin and belinostat were the most potent HDACIs with the mean 48 hour IC50 of 8.8 nM (range 1.7-2.7 nM) and 85 nM (range 36-136 nM), respectively. Cell viability was not affected by treatment with DEC or 5-AZA at 24 and 48 hours at concentrations as high as 20 μM. Reduction in viability was first demonstrated after 72 hours of exposure to DEC, with the mean IC50 of 14.8 μM (range 0.4 μM- >20uM). Simultaneous exposure of combinations of DEC plus romidepsin or DEC plus belinostat at their IC10, IC20, and IC50 produced marked synergy in all TCL derived cell lines. Simultaneous exposure of DEC plus romidepsin demonstrated the deepest synergy at 72 hours with synergy coefficients in the range of 0.3. Cells treated with the combination of DEC plus romidepsin also demonstrated significant induction of apoptosis as evaluated by annexinV/propridium iodide via FACS analysis and an increase in acetylated histone 3 by immunoblot. The in vivo activity of the combination of DEC plus belinostat was investigated in a xenograft model of CTCL using HH, the most resistant TCL derived cell line. Mice were treated with DEC 1.5 mg/kg (day 29, 33, 35, 37, 39, 41, 43) and/or belinostat 100 mg/kg (day 29-day 47). The combination mouse cohort demonstrated statistically significant tumor growth delay compared to DEC alone (p=0.002) and belinostat alone (p=0.001). The interaction of DEC and romidepsin was analyzed by GEP and methylation array. Interestingly, the baseline malignant phenotype seen in the CTCL cell-lines was reversed. A significant down-regulation of genes involved in biosynthetic pathways including protein and lipid synthesis, and a significant up-regulation of genes responsible for cell cycle arrest were seen. The vast majority (114/138; 92%) of genes modulated by the single agents were similarly modulated by the combination. However, the latter induced a further significant change in the transcriptome, affecting an additional 390 genes. Similarly, methylation array data was analyzed following treatment of these drugs alone and in combination. DEC induced de-methylation of 190 different gene regions corresponding to 175 genes and an additional 335 loci. Interestingly, when combined with romidepsin the number of demethylated gene regions decreased to 85 corresponding to 79 genes, 78 of which were common with DEC and 148 additional loci. The comparison of gene expression and methylation demonstrated a significant inverse relationship (R2 = 0.657) with genes found to be differentially expressed in GEP and methylation analysis. (Figure 1)Figure 1Summary of gene expression and methylation analysis.Figure 1. Summary of gene expression and methylation analysis. These data support the observation that DNMTIs in combination with HDACIs produces significant synergistic activity in models of TCL. Further evaluation of the mechanism of action with DNMTIs in combination with HDACIs is ongoing, and a clinical trial of the combination is now open. Disclosures: O'Connor: Celgene Pharmaceuticals: Consultancy; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Off Label Use: Hypomethylating Agents in T-cell lymphoma. Amengual:Acetylon Pharmacueticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Epigenetic Targeting with EZH2 and HDAC Inhibitors Is Synergistic in EZH2 Deregulated Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 839-839 ◽  
Author(s):  
Jennifer Kimberly Lue ◽  
Sathyen A Prabhu ◽  
Yuxuan Liu ◽  
Owen A. O'Connor ◽  
Jennifer E Amengual
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Lymphoma Cell ◽  
Advisory Committees ◽  
Genetics Research ◽  
Transcriptional Inhibition

Abstract EZH2 is critical in a process known as the Germinal Center (GC) reaction during which B-cells undergo somatic hypermutation and isotype switching in order to develop a large antibody repertoire. EZH2 is a histone methyltransferase serving as the catalytic subunit of the Polycomb Repression Complex 2 (PRC2), which is responsible for tri-methylation of histone 3 lysine 27 (H3K27), a mark of transcriptional repression. EZH2 recruits HDAC1/2 and DNMTs through its cofactor EED to further inhibit transcription. Mutations in EZH2 are found in 7-12% of FL and 22% of GC-DLBCL. EZH2 overexpression secondary to MYC and miRNA dysfunction has also been described. EZH2 also plays a role in T-cell differentiation and has been found in various T-cell malignancies. Histone acetyltransferases (HAT), notably CBP and p300, have also been implicated in B- and T-cell lymphomas and are mutated/deleted in 39% of GC-DLBCL and 41% of FL. Given the presence of EZH2 and HAT dysregulation in lymphoma, we evaluated the potential synergy of EZH2 and HDAC inhibitors co-treatment. Single agent activity for GSK126, an EZH2 inhibitor, and romidepsin, a pan-HDAC inhibitor, was established in a panel of lymphoma cell lines (GC-DLBCL, Non-GC DLBCL, MCL and T-Cell lymphoma, n=21). Cell lines with known EZH2 dysregulation (GC-DLBCL and ATLL) were more sensitive to EZH2 inhibitors as exhibited by lower half maximal effective concentration (EC50) after 6 day exposure (EC50 0.01-16 µM). There was no association between HAT mutation/deletion and romidepsin sensitivity. A panel of lymphoma cell lines was treated for 72 hr with GSK126 and romidepsin using concentrations represented by their EC30-50 (0.5-4.0 µM), and EC20-40 (1.0-4.0 nM), respectively. Synergy was assessed by Excess over Bliss (EOB), where EOB > 10 represents synergy. Simultaneous exposure to GSK126 and romidepsin in GC-DLBCL cell lines demonstrated potent synergy as represented by EOB > 30. Synergy was also present in ATLL cell lines (EOB 28), which are known to have EZH2 dysregulation, as well as non-GC DLBCL cell lines (EOB 47). Although these cell lines do not have EZH2 mutations, some possess relative EZH2 over-expression compared to other lymphomas. Evaluation of drug schedule using GSK126 pretreatment prior to romidepsin exposure did not impact synergy. Compared to single agent activity, the combination of GSK126 (2 µM) and romidepsin (1-4 nM) led to a more pronounced decrease in H3K27 tri-, di-, and mono-methylation and increased acetylation in 4 GC-DLBCL cell lines (OCI-LY7, Pfeiffer, SU-DHL-6, SU-DHL-10) at 24 or 48 hrs. The impact of the combination on the function of the PRC2 complex was assessed via co-immunoprecipation in these cell lines. The combination demonstrated dissociation of the PRC2 complex (EZH2, SUZ12, EED, and RbAp46/48) as compared to single agent exposure. Treatment with the combination also induced dissociation of HDAC2 and DNMT3L. In addition, we observed decreased protein expression of PRC2 complex members and increased p21/CDKN1A, which was more notable in the combination treatment as compared to single agent. This may be due to the removal of HDACs from the p21 transcriptional start site through the disruption of the PRC2 complex and direct inhibition of HDACs, thus leading to increase expression of p21. The combination also led to decreased nuclear localization of EZH2 and its cofactors. Apoptosis was confirmed by caspase 3 and PARP cleavage, and was more potently cleaved after exposure to the combination. Based on the findingthat HDAC2 dissociated from PRC2 complex after treatment with GSK126 and romidepsin, a selective HDAC1/2 inhibitor, ACY-957 (Acetylon Pharmaceuticals), was combined with GSK126 which demonstrated potent synergy in 4 GC-DLBCL cell lines (EOB 37). This data suggests that concomitant inhibition of EZH2 and HDAC is highly synergistic and leads to the dissociation of PRC2 complex. By releasing transcriptional inhibition key tumor suppressors and cell cycle regulators may be re-expressed. Potency of this epigenetic combination may be predicted by gene expression signatures for which RNA-seq libraries are currently in production. Reversing transcriptional inhibition using a combination of EZH2 inhibitors and HDAC inhibitors may lead to a potent treatment option for lymphomas dependent upon EZH2 and HAT activity. Figure 1 Figure 1. Disclosures O'Connor: Seattle Genetics: Research Funding; Spectrum: Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Mundipharma: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Celgene: Research Funding; Celgene: Research Funding. Amengual:Acetylon Pharmaceuticals: Research Funding; Bristol-Myers Squibb: Research Funding.

scholarly journals N-Cadherin Stabilizes β-Catenin and Promotes β-Catenin/TCF Transcriptional Activation and Cell Adhesion-Mediated Drug Resistance in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1572-1572
Author(s):  
Shiqiao Ye ◽  
Yu Chen ◽  
Bo Hu ◽  
Huayu Huang ◽  
Yuxiao Sun ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Board Of Directors ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Protein Levels ◽  
E Cadherin

Abstract Introduction: Inappropriate activation of Wnt/β-catenin signaling plays a role in some cancers. β-catenin (β-cat) levels in the cell can be regulated by a cadherin-mediated sequestration into membrane-bound and free cytosolic pools, with the later translocating to the nucleus and driving TCF-mediated transcriptional activity following Wnt signal transduction. While sequencing has shown that MM lacks the mutations that typically lead to constitutive β-cat activation seen in other cancers, we and others have demonstrated that Wnt/β-catenin signaling is nonetheless activated in MM and can regulate MM growth. The mechanism driving β-cat stabilization and activation in MM is unclear. E- and N-cadherin (N-cad) expression is elevated in MM compared to plasma cells from healthy donors. We hypothesized that that cadherins can regulate Wnt/β-catenin signaling in MM. Materials and Methods: We detected different forms of β-cat expression in a panel of human MM cell lines (HMCLs) and CD138 PC from MM patients by several approaches. Cadherin gain- or loss-of-function MM models were produced by expressing wild-type N-cad in MMS1 and ARP1 (lack endogenous N-cadherin expression) using a lentiviral system to create stable cell lines (N-Cad/MMS1 and N-cad/ARP1) and empty vector control (EV/MMS1, and EV-ARP1). We knocked down N-cadherin in the JJN3 cell line expressing high level of endogenous N-cadherin using shRNA specific for N-cad (shNcad/JJN3) or scrambled control shRNA (shCont/JJN3) by lentiviral-mediated transfection. We used a TCF reporter system to evaluate β-cat transcriptional activity as previously described. Results: We surveyed 25 HMCLs and CD138-selected plasma cells from 72 newly diagnosed MM for active β-cat with an antibody that specifically recognizes the unphosphorylated active form of β-cat. Higher levels of cytosolic and/or nuclear β-cat protein were seen in 13 of 25 (52%) HMCLs and 36 of 72 (50%) primary MM PC. Correlation of β-cat protein levels with global mRNA expression levels in primary PC revealed significant correlation with only one gene, CDH2 (N-cad). Remarkably, those primary MM with high β-cat levels but low CDH2 levels expressed high levels of E-cadherin/CHD1 mRNA. This posed the question of whether CDH2 is a direct target of TCF/β-cat transcriptional activity or whether high levels of CDH2 lead to increased levels of β-cat protein via sequestration. Both CDH2 mRNA and protein were correlated with β-cat protein but not β-cat mRNA in 23/25 HMCLs. Co-immunoprecipitation revealed that N-cad and β-cat complexes could be identified in HMCLs and primary MM. Consistent with N-cad-mediated stabilization of β-cat both total and unphosphorylated β-cat levels and TCF activity were significantly elevated in N-cad/MMS1 and N-Cad/ARP1 cells relative to controls. In contrast, shRNA mediated knockdown of N-cad led to a loss of both N-cad and β-cat protein levels and TCF-dependent transcription activity relative to controls. These findings provide evidence that β-cat/TCF signaling can be regulated by N-cad in MM. CDH2 mRNA is significantly elevated in the MS and HY subgroups of MM. To search for a potential mechanism of CDH2 transcriptional regulation in MS MM, we compared TCF activity and β-cat protein levels in MS versus non-MS HMCLs. TCF activity and active β-cat were elevated in MS versus non-MS forms of MM and B-cell lymphoma lacking N-cadherin. To determine if MMSET is required to up-regulate N-cad expression, we depleted the full-length MMSET protein in KMS11 cells. The results revealed a dramatic loss of total and unphosphorylated β-cat protein, but not mRNA, and loss of both CDH2 mRNA and protein relative to controls. These data suggest that MMSET can regulate the transcription of the CDH2 gene. MMS1 and ARP1 cells stably expressing N-cad exhibited enhanced adhesion to bone marrow stromal cells and decreased sensitivity to bortezomib (Bzb). In contrast, blocking N-cadherin-mediated adhesion by CDH2 shRNA increased sensitivity to Bzb. These results suggests that N-cad/β-cat complexes can contribute to adhesion-mediated drug resistance in MM. Conclusion: Taken together, these findings establish that β-cat is stabilized by N-cadherin-, and likely E-cadherin-, adhesins junction formation in MM. This in turn leads to an increased pool of β-cat that can drive TCF transcriptional activation as well participate in cadherin-mediated cell adhesion and drug resistance. Disclosures Davies: Amgen: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Morgan: BMS: Membership on an entity's Board of Directors or advisory committees; Jansen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Walker: Bristol Myers Squibb: Research Funding; Sanofi: Speakers Bureau.

scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

scholarly journals Activating KRAS, NRAS, and BRAF Mutants Enhance Proteasome Capacity and Reduce Endoplasmic Reticulum Stress in Multiple Myeloma, Thereby Promoting Plasma Cell Survival and Proteasome Inhibitor Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 406-406
Author(s):  
Fazal Shirazi ◽  
Richard J. Jones ◽  
Isere Kuiatse ◽  
Zuzana Berkova ◽  
Hua Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Transcription Factor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Research Funding ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Dominant Negative ◽  
Advisory Committees

Abstract Introduction: Multiple myeloma, a malignant proliferation of differentiated plasma cells, is the second most commonly diagnosed hematologic malignancy, and the number of cases may grow by almost 60% between 2010 and 2030. Recent therapeutic advances, including the use of proteasome inhibitors (PIs), have contributed to a doubling of the median overall survival in myeloma patients. This has been paralleled by an increased understanding of the mutational spectrum in this disease, which was first noted almost three decades ago to harbor KRAS and NRAS mutations. KRAS, NRAS, and BRAF mutations which induce p44/42 Mitogen-activated protein kinase (MAPK) signaling are found in about half of myeloma patients, and seem to contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms still remains elusive. Methods: ANBL-6 and U266 human-derived myeloma cell lines have endogenous wild-type (WT) KRAS, NRAS, and BRAF, and were used in this study. All cell lines were validated through The MD Anderson Cancer Center Characterized Cell Line Core Facility. We established lines stably expressing WT, constitutively active (CA)(G12V/G13D/Q61H), or dominant negative (DN)(S17N) KRAS and NRAS mutants, or V600E or DN BRAF. Cell viability was evaluated using the WST-1 tetrazolium reagent, while the chymotrypsin-, trypsin- and caspase-like activities were determined using fluorogenic substrates. Results: CA KRAS, NRAS, and BRAF mutants reduced the sensitivity of ANBL-6 and U266 cells to bortezomib and carfilzomib, while their DN variants sensitized cells to both PIs. This was associated with an induction by these CA mutants of the proteasome chymotrypsin-, trypsin- and caspase-like activities, while the DN variants reduced proteasome activity. These activity changes occurred in parallel with increased expression at both the mRNA and protein levels of catalytically active Proteasome subunit beta (PSMB)-8, PSMB9, and PSMB10, and of the proteasome assembly chaperone Proteasome maturation protein (POMP). Mechanistic studies showed that MAPK induction by the CA mutants caused activation of the ETS transcription factor (ELK1), which was found to have consensus binding sites in the promoters of PSMB8, PSMB9, PSMB10, and POMP. Notably, ELK1 suppression reduced PSMB8, PSMB9, PSMB10, and POMP expression, directly linking RAS/RAF/MAPK signaling to proteasome biology, and this suppression enhanced PI sensitivity. Inhibition of MAPK signaling with either the MAPK kinase (MEK) inhibitor selumetinib or the pan-RAF inhibitor TAK-632 showed synergistic activity with either bortezomib or carfilzomib that was more consistent in cell lines harboring CA mutants as opposed to the DN or WT constructs. Combination regimens of selumetinib or TAK-632 with either bortezomib or carfilzomib induced greater inhibition of the proteasome chymotrypsin-, trypsin- and caspase-like activities than the PIs as single agents. Finally, CA KRAS, NRAS, and BRAF mutants reduced expression levels of genes and proteins involved in the unfolded protein response (UPR), including Activating transcription factor (ATF)-4, -5, and -6, as well as C/EBP homologous protein transcription factor (CHOP) and the spliced variant of X-box binding protein 1 (XBP1s). In contrast, their dominant negative counterparts enhanced expression of the UPR effectors, consistent with an increase in endoplasmic reticulum (ER) stress. Conclusion: Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue that these mutations promote plasma cell survival by reducing cellular stress, thereby distancing myeloma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma. Disclosures Lee: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies Corporation: Consultancy; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai Biopharmaceuticals: Consultancy; Takeda Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees. Dick:Takeda Oncology: Employment, Equity Ownership. Chattopadhyay:Takeda Oncology: Employment. Orlowski:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Poseida: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Mao Yu Peng ◽  
Yasmin Abaza ◽  
Martina Mcdermott ◽  
Monica Mead ◽  
Dennis J. Slamon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Synergistic Effect ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
Single Agent ◽  
Therapeutic Strategies ◽  
Private Company ◽  
Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates &gt;85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

scholarly journals Simultaneous Kinase Inhibition with Ibrutinib and BCL2 Inhibition with Venetoclax As a Therapeutic Strategy for Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3950-3950
Author(s):  
Christopher A. Eide ◽  
Stephen E Kurtz ◽  
Andy Kaempf ◽  
Nicola Long ◽  
Jessica Leonard ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership

Background: In patients with acute lymphoblastic leukemia (ALL), patient outcomes vary considerably by patient age group, specific genetic subtypes, and treatment regimen. Large-scale sequencing efforts have uncovered a spectrum of mutations and gene fusions in ALL, suggesting that combinations of agents will be required to treat these diseases effectively. Previous preclinical studies have shown efficacy of the BCL2 inhibitor venetoclax alone or in combination in ALL cells (Chonghaile et al., Can Disc 2014; Leonard et al, STM 2018), and the multi-kinase inhibitor ibrutinib (approved for patients with chonic lymphoblastic leukemia (CLL)) has also shown potent activity in subsets of ALL (Kim et al., Blood 2017). However, the combination of ibrutinib and venetoclax has not been evaluated to date in patients with ALL. Methods: We used a functional ex vivo screening assay to evaluate the potential efficacy of the combination of ibrutinib and venetoclax (IBR+VEN) across a large cohort (n=808) of patient specimens representing a broad range of hematologic malignancies. Primary mononuclear cells isolated from leukemia patients were plated in the presence of graded concentrations of venetoclax, ibrutinib, or the combination of both FDA-approved drugs. IC50 and AUC values were derived from probit-based regression for each response curve. A panel of clinical labs, treatment information, and genetic features for tested ALL patient specimens was collated from chart review. Single and combination drug treatment sensitivity were compared within groups by Friedman test, across groups by Mann-Whitney test, and with continuous variables by Spearman rank correlation. Results: Consistent with clinical data and previous literature, IBR+VEN was highly effective in CLL specimens ex vivo (median IC50=0.015 µM). Intriguingly, among specimens from 100 unique ALL patients, we also observed that IBR+VEN demonstrated significantly enhanced efficacy by AUC and IC50 compared to either single agent (p<0.001; median IC50=0.018 µM). In contrast, evaluation of this combination on primary mononuclear cells from two healthy donors showed little to no sensitivity. Breakdown of combination sensitivity (as measured by AUC) by a variety of clinical and genetic features revealed no associations with gender or specimen type. Among continuous variables tested, age was modestly correlated with combination AUC (Spearman r = 0.26) and increased blasts in the bone marrow were associated with increased sensitivity to the combination (Spearman r = -0.41; p = 0.0068). More broadly, specimens from patients with B-cell precursor disease (B-ALL) were more sensitive to IBR+VEN than those with T-cell precursor leukemia (T-ALL) (p = 0.0063). Within the B-ALL patient samples, those harboring the BCR-ABL1 fusion were significantly less sensitive to IBR+VEN than other subtypes of B-ALL (p = 0.0031). Within the T-ALL subset, there was a trend toward reduced sensitivity in patients with evidence of mutations in NOTCH1, though statistical significance was not reached. Evaluation of the combination using an expanded 7x7 concentration matrix in human ALL cell lines revealed varying degrees of sensitivity. For example, IBR+VEN showed enhanced efficacy in RCH-ACV B-ALL cells and showed synergy for the majority of drug-pair concentrations by the highest single agent (HSA) method (ibrutinib, venetoclax, and combination IC50: 0.60, 3.4, and 0.28 uM, respectively). Conclusion: Our findings suggest that the IBR+VEN combination, currently approved for patients with CLL, also demonstrates impressive efficacy against primary leukemia cells from ALL patients, warranting further investigation as a treatment strategy in the clinic to continue to improve outcomes for patients. Disclosures Leonard: Amgen: Research Funding. Druker:Cepheid: Consultancy, Honoraria; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Gilead Sciences: Other: former member of Scientific Advisory Board; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; CureOne: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees. Tyner:Petra: Research Funding; Agios: Research Funding; Array: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; AstraZeneca: Research Funding; Seattle Genetics: Research Funding; Array: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Syros: Research Funding; Takeda: Research Funding; Petra: Research Funding; Agios: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Gilead: Research Funding; Incyte: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Janssen: Research Funding; Genentech: Research Funding.

The Aurora A Kinase Inhibitor, Alisertib, Has Broad Activity In Nonclinical Models Of T-Cell Lymphoma and Is Highly Synergistic With Romidepsin, But Not With Pralatrexate Or The Proteasome Inhibitor, Ixazomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5141-5141 ◽  
Author(s):  
Kelly Zullo ◽  
Luigi Scotto ◽  
Jennifer E. Amengual ◽  
Owen A. O'Connor
Keyword(s):  
T Cell ◽  
Board Of Directors ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Time Effect ◽  
T Cell Lymphoma ◽  
Aurora A Kinase ◽  
Advisory Committees

Abstract Aurora A kinase (AAK), a serine-threonine protein kinase, regulates mitotic entry, spindle formation, and cytokinesis. Alisertib is a selective AAK investigational inhibitor with demonstrated clinical activity in acute myeloid leukemia, peripheral t-cell lymphoma (PTCL), DLBCL and other heme-lymphatic cancers. Here we report the cytotoxicity and apoptotic effect of Alisertib  in a panel of T-cell-derived lymphoma cell-lines (TCL) (CTCL, HTLV+, T-ALL) and B-cell lymphoma cell-lines (DLBCL-ABC, DLBCL-GCB, MCL) alone and in combination with romidepsin, pralatrexate (PDX) and ixazomib, a  proteasome inhibitor. Single agent concentration and time effect relationships were generated for 8 TCL, 4 DLBCL (2 ABC, 2 GCB) and 4 MCL cell-lines. The mean IC50 of alisertib in TCL was 350 nM (range 100-1000nM) and in B-cell lymphoma lines (DLBCL, MCL) was 200 nM (range 20-300 nM) at 48 hours, measured by growth inhibition. In all cell lines evaluated, there was a consistent 2-log fold decrease in IC50 values at 72 hours.  Combination studies evaluating synergy were performed testing schedule, concentration, and time effect relationships. Interestingly, simultaneous exposure of combined alisertib and romidepsin at their IC10, IC20, and IC30 demonstrated marked synergy in TCL. Deepest synergy was observed at 72 hours with synergy coefficients ranging from 0.2 to 0.7. This synergistic interaction was restricted to the TCL cell-lines, with no benefit demonstrated in DLBCL or MCL cell lines. (Table 1) Similarly, alisertib did not demonstrate synergy in TCL, DLBCL or MCL cell lines at any concentration, combination, or time schedule  with PDX  or ixazomib (simultaneous incubation of alisertib + PDX, 24 hour pre-exposure alisertib followed by PDX, 24 hour pre-exposure PDX followed by alisertib; simultaneous incubation of alisertib + ixazomib).Table 1Synergy Coefficients of Alisertib in Combination with Romidepsin at 72 Hours.Combination DND41(T-ALL) J.CAM1.6(T-ALL) HH(CTCL) H9(CTCL) C5MJ(HTLV+) Romidepsin [IC10-20] +Alisertib 50 nM 0.96 0.81 1.05 1.1 1.53 Romidepsin +Alisertib 100nM 0.51 0.56 0.68 0.66 0.58 Romidepsin +  Alisertib 1000nM 0.40 0.20 0.40 0.46 0.70 1 1=additive; <1=synergistic; >1= subadditive Evidence for apoptosis was confirmed for alisertib in combination with romidepsin in the TCL cell-line, H9 after 48 hours of exposure by increased caspase 3 and PARP cleavage,  acetylated H3 expression, and decreased Cyclin B1, P27, and BCL2 expression; suggesting that cell death occurred through apoptosis.  AnnexinV/propridium iodide via FACS analysis confirmed induction of apoptosis. These data support the observation that alisertib produces broad single-agent activity in models of TCL and demonstrates marked synergy with romidepsin. Interestingly this effect is restricted to TCL and there is no synergistic effect with ixazomib or PDX in TCL.  Further evaluation of the mechanism of action with alisertib in combination with romidepsin as well as in vivo modeling of these combinations is ongoing. Disclosures: Off Label Use: Aurora kinase inhibitors are not approved for T-Cell lymphoma. Amengual:Acetylon Pharmaceuticals, INC: Membership on an entity’s Board of Directors or advisory committees, Research Funding. O'Connor:Millennium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Therapeutics: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy.

Synergistic Anti-Myeloma Activity of a Proteasome Inhibitor Marizomib and IMiD® Immunomodulatory Drug Pomalidomide

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2099-2099
Author(s):  
Deepika Sharma Das ◽  
Durgadevi Ravillah ◽  
Arghya Ray ◽  
Yan Song ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Response To Treatment ◽  
Low Doses ◽  
Advisory Committees ◽  
Healthy Donors

Abstract Background and Rationale: Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that a novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). We also showed that marizomib triggers synergistic anti-MM activity in combination with lenalidomide (Chauhan et al., Blood 2010, 115:834-45). Pomalidomide, like lenalidomide, is an analogue of thalidomide with potent immunomodulatory activity, and has been approved by FDA for treatment of RRMM patients who have received at least two prior therapies including lenalidomide and bortezomib and showed disease progression on or within 60 days of completion of the last therapy. Approval of treatment is based on progression-free survival. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide. Materials and Methods:MM celllines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of marizomib and pomalidomide. Cell viability, apoptosis, and migration assays were performed using WST/MTT, Annexin V staining, and Transwell Inserts, respectively. Synergistic/additive anti-MM activity was analyzed by isobologram analysisusing “CalcuSyn” software program. Proteasome activity was measured, as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. MM.1S-tumor-bearing mice were treated with vehicle control, marizomib, pomalidomide or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student’s t test. Pomalidomide was purchased from Selleck chemicals, USA; and marizomib was obtained from Triphase Inc., USA. Results: MM cell lines (MM.1S, MM.1R, INA-6, RPMI-8226, Dox-40, U266, LR5, ANBL6.WT, and ANBL6.BR) and primary patient MM cells were pretreated with DMSO control or with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by assessment of cell viability. A significant decrease in viability of all cell lines and patient cells was observed in response to treatment with combined low doses of marizomib and pomalidomide, compared with either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to conventional (dex, doxorubicin, melphalan) and novel (bortezomib) therapies. No significant decrease in viability of PBMCs from normal healthy donors was observed in response to treatment with combined low doses of marizomib and pomalidomide, suggesting selective anti-MM activity and a favorable therapeutic index for this combination regimen. Furthermore, marizomib plus pomalidomide inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies showed that marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) downregulation of Cereblon, IRF4, c-Myc, and Mcl-1; and 3) enhanced inhibition of chymotrypsin-like, caspase-like and trypsin-like proteasome activities versus single agent alone. Furthermore, combined low doses of marizomib and pomalidomide blocked migration of MM cells and angiogenesis. In vivo studies using a subcutaneous human MM xenograft models show that combined low doses of marizomib and pomalidomide are well tolerated, inhibit tumor growth, and prolong survival. Conclusion: Our preclinical studies in MM disease models support a clinical trial of combined marizomib and pomalidomide to improve outcome in patients with relapsed and refractory MM. Disclosures Richardson: Oncopeptides AB: 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. Trikha:Triphase Accelerator: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

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