Targeting Sp1 Transactivation In Waldenstrom's Macroglobulinemia: a Novel Therapeutic Option

Abstract Abstract 120 The transcription factor Sp1 transactivates expression of genes containing proximal GC/GT-rich promoter elements controlling cell differentiation, cell cycle and apoptosis affecting growth and survival of tumor cells. Based on previous observation that key multiple myeloma (MM) cell growth and survival genes such as NF-kB p65, IGF-IR, VEGF, and IL-6 are controlled by Sp proteins, we have previously investigated and observed high Sp1 expression and activity in MM cells and confirmed its role in MM by Sp1 knock down using both siRNA and lentiviral shRNA constructs specific for Sp1. We further evaluated the role of Sp-1 in WM and observed high nuclear Sp1 protein expression along with increased Sp1 activity in WM cells compared to normal peripheral blood mononuclear cells (PBMC). Moreover, adhesion of WM cells to bone marrow stromal cells (BMSC) further induces Sp1 activity in WM cells. Based on these data, we have investigated the anti-WM activity of Terameprocol (TMP), a small molecule suitable for clinical application,which specifically competes with Sp1-specific DNA binding domains within gene promoter regions. It disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity without direct effect on its expression. We have confirmed inhibition of both basal and BMSC-induced binding and transcriptional activity of Sp1 in WM cells using an ELISA assay specific for measuring Sp1 binding activity and using Sp1 sensitive luciferase reporter plasmid. TMP treatment did not affect Sp1 protein levels. Importantly, TMP significantly inhibited WM cell growth in a dose-dependent fashion (IC50 between 5–20 μ M at 24 hours) and was able to overcome the protective effects of BMSCs. TMP activates the mitochondrial apoptotic pathway via induction of caspase-3, -9 and -7 and PARP cleavage but without caspase-8 activation. TMP treatment also led to downregulation of expression of survivin, a known anti-apoptotic gene transcriptionally regulated by Sp1. We have also confirmed in vivo activity of TMP in a murine xenograft model of MM. Finally based on the data suggesting that both dexamethasone and revlimid increase Sp1 activity, we have combined TMP with these agents and observed synergistic activity on cell growth and survival. In conclusion, our results demonstrate Sp1 as an important transcription factor in WM and provides preclinical rationale for clinical development of TMP as a specific Sp1 inhibitor alone and in combination with conventional and novel agents in WM. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Treon:Millennium Pharmaceuticals, Genentech BiOncology, Biogen IDEC, Celgene, Novartis, Cephalon: Consultancy, Honoraria, Research Funding; Celgene Corporation: Research Funding; Novartis Corporation: Research Funding; Genentech: Consultancy, Research Funding. Munshi:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

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Biology and Therapeutic Targeting of Sp1 Transactivation In Myeloma

Blood ◽

10.1182/blood.v116.21.134.134 ◽

2010 ◽

Vol 116 (21) ◽

pp. 134-134

Author(s):

Mariateresa Fulciniti ◽

Samir B. Amin ◽

Puru Nanjappa ◽

Scott J Rodig ◽

Teru Hideshima ◽

...

Keyword(s):

Cell Growth ◽

Board Of Directors ◽

Luciferase Reporter ◽

Parp Cleavage ◽

Specific Inhibition ◽

Advisory Committees ◽

Growth And Survival ◽

Synergistic Cytotoxicity

Abstract Abstract 134 Alteration in expression and function of transcription factors has been frequently associated with neoplastic transformation. We here provide both experimental and clinical evidence that Sp1, a transcription factor that controls number of cellular processes, plays an important regulatory role in MM cell growth and survival. Although Sp1 is ubiquitously expressed, its nuclear localization observed in MM is functionally both relevant and important. We have confirmed high Sp1 activity in MM cells both by demonstrating increased DNA binding as well as increased Sp1-responsive promoter activity measured by luciferase reporter assay. MM cell-BMSC interaction led to Sp1 activation which was completely abrogated by the ERK pathway inhibitor U0126 but not by the AKT inhibitor LY29004. Furthermore, using both SiRNA and ShRNA mediated Sp1 knock-down, we have confirmed the growth and survival effects of Sp1 on MM cell growth. Using gene expression profile of MM cells from 172 uniformly treated patients, we have further confirmed these in vitro results by observing that overexpression of Sp1-related genes, including HIF-1a, HDAC1 and MAPK genes, correlate with poor prognosis in MM. This clinical correlation further suggests the role of Sp1 in MM biology, providing the rationale to preclinically target Sp1 in MM. We have investigated TMP, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA) which disrupts the interaction between Sp1 and GC-rich motifs inhibiting Sp1 activity. We have previously confirmed specific inhibition of both Sp1 binding and transcriptional activity in MM cells by TMP, including in the context of MM-BMSC interaction. Along with inhibition of Sp1 activity, we observed both in vitro and in vivo in murine models of human myeloma, anti-myeloma effect of TMP. Importantly, there was no significant synergistic effect when MM cells transfected with Sp1 siRNA were treated with TMP, confirming specificity of TMP's mechanism of action. We have further observed activation of the mitochondrial apoptotic pathway by TMP via activation of caspase-3, -9 and -7 and PARP cleavage while caspase-8 was not activated suggesting possible synergism with activators of alternate apoptotic pathways. We have also observed that lenalidomide and dexamethasone upregulate Sp1 activity, suggesting that Sp1 may be a common resistance mechanism. We have confirmed that the increased Sp1 activity by lenalidomide or dexamethasone is abrogated by TMP and the combination provides synergistic cytotoxicity, in MM cell lines as well as primary MM cells. In conclusion, we report significant role of Sp1 in myeloma cell growth, survival and drug resistance with its influence on clinical outcome in MM. Our results suggest that specific inhibition of Sp1 activity may be an important therapeutic target in MM. Disclosures: Avet-Loiseau: Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

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Functional and Clinical Impact of Splicing Factor Dysregulation in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.726.726 ◽

2015 ◽

Vol 126 (23) ◽

pp. 726-726 ◽

Cited By ~ 1

Author(s):

Mariateresa Fulciniti ◽

Mehmet Kemal Samur ◽

Naim Ur Rashid ◽

Rajya Lakshmi Bandi ◽

Manoj Bhasin ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Growth ◽

Board Of Directors ◽

Plasma Cells ◽

Splicing Factor ◽

Alternate Splicing ◽

Advisory Committees ◽

Growth And Survival ◽

Cell Growth And Survival

Abstract Transcriptome modifiers such as alternative pre-mRNA splicing (AS), long non-coding RNA and microRNA (miRNA) need to be considered in order to provide a more accurate genomic framework for clinical correlation, as well as for high value therapeutic target discovery. Aberrant splicing of numerous genes has been reported in other malignancies, including a small number of genes reported in MM. We have evaluated AS in MM by analyzing clinically annotated high throughput RNA-seq data from 410 newly-diagnosed patients and 18 normal donor plasma cells. We observed a profound and significant AS in MM with over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. Importantly, unsupervised analysis identified clinically relevant MM subgroups with high and low splicing index respectively and showed significant impact of alternate splicing on overall clinical outcome. Based on these data, we next focused on understanding the molecular mechanisms driving aberrant alternate splicing in myeloma. Several studies provide evidence that an abnormally expressed splicing factor (SF) can have oncogenic properties by impacting alternative splicing of cancer-associated genes. We detected dysregulated expression of several SFs, including SF3B1, Fox2, SRSF1, NONO, in patients with MM compared to normal plasma cells with impact on outcome, highlighting for the first time the prognostic significance of splicing related factors in myeloma. We further observed that overexpression of some of these SFs increased cell proliferation, enhanced anchorage independent growth in semi-solid medium, and affected tumorigenic potential. We have further investigated role of Serine/Arginine Splicing Factor 1 (SRSF1) in MM by gain of- and loss of- function studies. Enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells. Conversely, transient or stable downregulation of SRSF1 with specific siRNA and shRNAs respectively, significantly inhibited MM cell proliferation and cell survival. We have also investigated a small molecular inhibitor of SRSF1 (TG003) and observed inhibition of MM cell growth and survival. The impact of this inhibitor on allelic isoforms of specific gene targets is undergoing. To dissect the mechanisms involved in the SRSF1-mediated MM growth induction, we used SRSF1 mutants lacking either of the two RNA-recognition motifs (ΔRRM1 or ΔRRM2 mutants) or the serine/argine-rich C-terminal domain (ΔRS mutant) involved in protein-protein interactions, subcellular localization, and recruitment of spliceosome components. We also used a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. We surprisingly found that only NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation. Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of E2F1, a transcription factor with significant impact on MM cell growth and survival. A significant reduction in SRSF1 at mRNA and protein levels was observed after E2F1 and/or E2F1 heterodimerization partner Dp1 gene silencing. Moreover, peptide-based strategy to abrogate interaction between Dp1-E2F1 led to decreased SRSF1 expression levels. These results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM. The study highlights the need to further understand the splicing pattern in myeloma and also supports the emerging concept that splicing programs, together with transcriptional programs participate in the altered cellular function during tumor initiation and progression. Disclosures Munshi: onyx: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees.

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Targeting Brouton's Tyrosine Kinase with PCI-32765 Blocks Growth and Survival of Multiple Myeloma and Waldenström Macroglobulinemia Via Potent Inhibition of Osteoclastogenesis, Cytokines/Chemokine Secretion, and Myeloma Stem-Like Cells in the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v118.21.883.883 ◽

2011 ◽

Vol 118 (21) ◽

pp. 883-883

Author(s):

Yu-Tzu Tai ◽

Betty Y Chang ◽

Sun-Young Kong ◽

Mariateresa Fulciniti ◽

Guang Yang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tyrosine Kinase ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Growth And Survival ◽

Equity Ownership ◽

Trap Staining

Abstract Abstract 883 Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in regulating osteoclastogenesis. Although Btk is critical in B cell maturation and myeloid function, it has not been characterized in plasma cell malignancies including multiple myeloma (MM) and Waldenström Macroglobulinemia (WM). We here investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) microenvironment, as well as on MM and WM cancer cells. We further define molecular targets of Btk signaling cascade in OCs and MM in the BM milieu. In CD14+ OC precursor cells, RANKL and M-CSF stimulate phosphorylation of Btk in a time-dependent fashion; conversely, PCI-32765 abrogates RANKL/M-CSF-induced activation of Btk and downstream PLCγ2. Importantly, PCI-32765 decreased number of multinucleated OC (>3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and the secretion of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It increased size of OCs and number of nuclei per OC, with significantly defective bone resorption activity as evidenced by diminished pit formation on dentine slices. Moreover, lack of effect of Dexamethasone on OC activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 significantly reduced cytokine and chemokine secretion from OC cultures, including MIP1α, MIP1β, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1–0.48 nM). It potently decreased IL-6, SDF-1, MIP1α, MIP1β, and M-CSF in CD138-negative cell cultures from active MM patients, associated with decreased TRAP staining in a dose-dependent manner. In MM and WM cells, immunoblotting analysis confirmed a higher Btk expression in CD138+ cells from majority of MM patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated a higher expression of Btk and its downstream signaling components in WM cells than in CD19+ normal bone marrow cells. PCI-32765 significantly inhibits SDF-1-induced adhesion and migration of MM cells. It further blocked cytokine expression (MIP1a, MIP-1β) at mRNA level in MM and WM tumor cells, correlated with inhibition of Btk-mediated pPLCγ2, pERK and NF-kB activation. Importantly, PCI-32765 inhibited growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs in IL-6-dependent INA6 and ANBL6 MM cells. Furthermore, myeloma stem-like cells express Btk and PCI-32765 (10–100 nM) blocks their abilities to form colonies from MM patients (n=5). In contrast, PCI-32765 has no adverse effects on Btk-negative BMSCs and OBs, as well as Btk-expressing dendritic cells. Finally, oral administration of PCI-32765 (12 mg/kg) in mice significantly suppresses MM cell growth (p< 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results provide compelling evidence to target Btk in the BM microenvironment against MM and WM., strongly supporting clinical trials of PCI-32765 to improve patient outcome in MM and WM. Disclosures: Chang: Pharmacyclics Inc: Employment. Buggy:Pharmacyclics, Inc.: Employment, Equity Ownership. Elias:Pharmacyclics Inc: Consultancy. Treon:Millennium: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Genentech: Honoraria. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: 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 Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol-Myers Squibb: Consultancy; Actelion: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Novel BET Protein Degrader-Based Combinations Exert Lethality Against Human AML Resistant to BET Inhibitors Due to Increased Activity of β-Catenin-TCF7L2- MYC Axis

Blood ◽

10.1182/blood-2018-177 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 177-177

Author(s):

Dyana T. Saenz ◽

Warren Fiskus ◽

Taghi Manshouri ◽

David N Saenz ◽

Raffaella Soldi ◽

...

Keyword(s):

Transcription Factor ◽

Progenitor Cells ◽

Board Of Directors ◽

Research Funding ◽

Genetic Alterations ◽

Advisory Committees ◽

Lethal Effects ◽

Bet Inhibitors ◽

Nuclear Levels ◽

And Control

Abstract Bromodomain and extra-terminal protein (BETP) inhibitors (BETis) disrupt the chromatin binding and activity of the BETP BRD4 in facilitating RNA pol II-mediated mRNA transcription, thereby depleting levels of active oncoproteins including c-Myc, CDK6, BCL2, PIM1 and MCL1. BETi treatment also increases protein levels of p21, p27 and HEXIM1, thereby causing growth inhibition and apoptosis of AML blast progenitor cells (BPCs), including post-MPN, secondary AML (sAML) BPCs. Treatment with BETi (e.g., OTX015) has been shown to reduce AML burden and induce clinical remissions. However, BETi-refractory AML develops uniformly. Previous reports utilizing mouse AML models have highlighted that persister-resistance to BETi (BETi-P/R) in AML stem progenitor cells is observed despite BETi treatment and reduction of BRD4 occupancy on the chromatin. This is mediated by re-expression of c-Myc due to transcriptional activity of WNT-β-catenin. In the present studies, we developed human sAML models of BETi-P/R to elucidate the mechanisms and develop targeted therapies against BETi-P/R sAML BPCs. Utilizing human sAML control (parental) SET2 and HEL92.1.7 cells and subjecting them to at least 10 exposures to 1.0 µM of the BETi OTX015 for 48 hours followed by full recovery, we first generated the BETi-P/R SET2-P/R and HEL-P/R cells. These cells were > 10-fold resistant to OTX015 and exhibited cross-resistance to other BETis, including JQ1 and ABBV-075. As compared to the control sAML cells, SET2-P/R and HEL-P/R cells neither exhibited additional genetic alterations by NextGen whole-exome sequencing, nor showed altered levels of TRIM33, SPOP or phosphorylated BRD4 (previously described mechanisms of BETi-resistance). In contrast, compared to the control, SET2-P/R and HEL-P/R cells demonstrated significantly higher nuclear levels and binding of β-catenin to the transcription factor TCF7L2 (TCF4) and TBL1X (TBL1), associated with increased expression of TCF4 targets, including c-Myc, Cyclin D1, TERT and Survivin. ATAC-Seq and ChIP-Seq (H3K27Ac mark) analyses showed significant gain of peaks and active enhancers in HEL-P/R over HEL92.1.7 cells, including enrichment of the STAT5, MYC, PU.1, GATA2 and MYB transcription factor binding sites, as well as newly gained peaks in the enhancers of JAK1/2, RUNX1, PU.1, MYC, BCL2L1 and CTNNB1. RNA-Seq analysis showed significant increase/decrease in mRNA expressions (340/247), with increased expression of gene-sets involving MYC/MAX, STAT5, NFkB and TCF4 targets. QPCR and Western analyses confirmed significant perturbation in gene expressions, with increase in TCF4, c-Myc, Survivin and PIM1 in HEL-P/R over HEL92.1.7 cells. Consistent with the finding that shRNA-mediated knockdown of BRD4 exerted similar lethal effects in BETi-P/R versus control cells, we also discovered that BETP-PROTAC (proteolysis targeting chimera) ARV-771 (Arvinas, Inc.) that degraded BRD4/3/2 was equipotent in inducing apoptosis of BETi-P/R and control sAML cells. Also, consistent with increased nuclear levels and binding (utilizing confocal microscopy) of β-catenin with TBL1 and TCF4 in BETi-P/R sAML BPCs, β-catenin inhibitor BC2059 (Beta-Cat Pharma), which disrupts the binding of nuclear β-catenin with TBL1 and TCF4 and depletes β-catenin levels, exerted similar lethal effects in BETi-P/R sAML and control sAML cells. Consistent with these findings, we also determined that co-treatment with ARV-771 and BC2059 exerted synergistic in vitro lethality against BETi-P/R sAML BPCs (combination indices < 1.0), which was associated with greater reduction in levels of c-Myc, TCF4, Survivin, CDK6, PIM1 and Bcl-xL. Co-treatment with ARV-771 and BC2059 was also synergistically lethal against 12 patient-derived samples of CD34+ sAML BPCs. Notably, compared to treatment with each agent alone or vehicle control, in vivo treatment with ARV-771 (30 mg/kg SQ daily x 5, per week) and BC2059 (30 mg/kg IP BIW per week) for 3 weeks, significantly reduced the sAML burden and improved survival of the NSG mice engrafted with luciferase-transduced HEL-P/R cells (p < 0.01). These findings demonstrate that increased levels and activity of β-catenin-TCF7L2-MYC axis is mechanistically responsible for BETi-P/R, and co-targeting with BETP degrader and β-catenin-TCF4 inhibitor is synergistically lethal against BETi-P/R sAML BPCs. Disclosures Soldi: Beta Cat Pharma: Employment. Bose:Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Blueprint Medicines Corporation: Research Funding; Pfizer, Inc.: Research Funding; Constellation Pharmaceuticals: Research Funding; CTI BioPharma: Research Funding; Incyte Corporation: Honoraria, Research Funding. Kadia:BMS: Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Celgene: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy. DiNardo:Abbvie: Honoraria; Medimmune: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Agios: Consultancy. Horrigan:Beta Cat Pharma: Employment. Khoury:Stemline Therapeutics: Research Funding. Verstovsek:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy.

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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 ◽

10.1182/blood-2018-99-117577 ◽

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.

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FLT3 and LSD1 Inhibitor Combinations Synergistically Repress Growth of FLT3-Mutant Acute Myeloid Leukemia Via Blockage of MYC Function

Blood ◽

10.1182/blood-2020-143029 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Daniel J. Coleman ◽

Brittany M. Smith ◽

Cody Coblentz ◽

Rowan L. Callahan ◽

Jake VanCampen ◽

...

Keyword(s):

Transcription Factor ◽

Board Of Directors ◽

Transcriptional Activation ◽

Myeloid Leukemia ◽

Research Funding ◽

Target Genes ◽

Publicly Traded ◽

Private Company ◽

Advisory Committees ◽

Flt3 Inhibitor

Internal Tandem Duplication mutations of Fms Related Receptor Tyrosine Kinase 3 (FLT3), known as FLT3-ITD mutations, are associated with poor prognosis in Acute Myeloid Leukemia (AML). The clinical efficacy of inhibiting FLT3 in AML is limited by the rapid development of drug resistance and relapse, underscoring a need for more potent and durable treatment strategies. The early persistence of leukemic blasts during FLT3 inhibition is a key driver of resistance. We find that in combination, inhibitors of Lysine Specific Demethylase 1 (LSD1) potentiate the activity of FLT3 inhibitors, driving synergistic cell death. This novel therapeutic approach has the potential to drive deeper therapeutic responses in FLT3-Mutant AML, delaying or preventing the development of resistance. LSD1 is a dynamic DNA-associated protein that functions as a chromatin modifier and transcription factor. LSD1 removes methylation on both lysine 4 of histone H3 (H3K4), associated with transcriptional activation, and lysine 9 (H3K9), associated with transcriptional repression. Additionally, LSD1 has been reported to function as a transcription factor independent of its catalytic demethylase function. LSD1 inhibition reduces cell proliferation in several cancer types. In AML specifically, inhibition of LSD1 has been reported to activate enhancers associated with genes that promote differentiation. We hypothesized that combining LSD1 inhibition with FLT3 inhibition in FLT3-ITD AML would result in synergistic effects on cell viability through reactivating differentiation pathways and more strongly blocking proliferation. In this study, we aimed to examine the efficacy, transcriptional effects, and changes in chromatin dynamics when combining LSD1 inhibition with FLT3 inhibition in a FLT3-ITD mutant cell line and patient samples. We used matrix combination screening to determine that combining the FLT3 inhibitor Quizartinib with LSD1 inhibitors (GSK-2879552 or ORY-1001) synergistically represses cell viability in the FLT3-ITD mutant MOLM-13 cell line and in multiple primary AML samples. RNA-seq followed by Gene Set Enrichment Analysis revealed that combining LSD1 and FLT3 inhibition synergistically represses target genes of the oncogenic transcription factor MYC. This finding was corroborated through high-throughput genome-wide profiling of histone marks, using the recently developed technique Cleavage Under Targets and Tagmentation (CUT&Tag). Specifically, we discovered several promoter regions in which acetylation of lysine 27 of Histone H3 (H3K27Ac), associated with transcriptional activation, was repressed by combining LSD1 and FLT3 inhibition. The genes associated with these regions were strongly enriched for known MYC target genes. Through additional genomic profiling methods including ChIP-seq and ATAC-seq, we have established potential roles for several DNA-binding transcription factors including CEBPA, RUNX1, STAT5, and LSD1 itself, that may mediate repression of MYC function resulting from combining LSD1 and FLT3 inhibition. Together, our work establishes LSD1 and FLT3 inhibitor combinations as a promising treatment strategy in FLT3-ITD AML. Importantly, this study identifies combined FLT3 and LSD1 inhibition as an effective strategy to indirectly target MYC function, as MYC is often referred to as an "undruggable" target. Furthermore, it has the potential to drive deeper molecular responses in FLT3-mutant AML, decreasing the likelihood of treatment resistance. Disclosures Druker: Bristol-Myers Squibb: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Patient True Talks: Consultancy; Oregon Health & Science University: Patents & Royalties; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Leukemia & Lymphoma Society: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding. Maxson:Gilead Sciences: Research Funding; Ionis Pharmaceuticals: Other: Joint oversight committee for a collaboration between OHSU and Ionis Pharmaceuticals.

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Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells

Blood ◽

10.1182/blood.v116.21.791.791 ◽

2010 ◽

Vol 116 (21) ◽

pp. 791-791 ◽

Cited By ~ 1

Author(s):

Diana Cirstea ◽

Teru Hideshima ◽

Loredana Santo ◽

Samantha Pozzi ◽

Sonia Vallet ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Drug Resistance ◽

Cell Growth ◽

Cell Survival ◽

Board Of Directors ◽

Research Funding ◽

Kinase Inhibitor ◽

Advisory Committees ◽

Mtor Kinase

Abstract Abstract 791 Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures: Guichard: AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

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Phase I Trial of Everolimus and Rituximab or Everolimus, Bortezomib and Rituximab in Relapsed or Relapsed/Refractory Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v118.21.2705.2705 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2705-2705 ◽

Cited By ~ 1

Author(s):

Irene M. Ghobrial ◽

Erica N Boswell ◽

Ranjit Banwait ◽

Tiffany Poon ◽

Amanda Donovan ◽

...

Keyword(s):

Phase I ◽

Board Of Directors ◽

Research Funding ◽

Single Agent ◽

Recommended Dose ◽

Synergistic Activity ◽

Advisory Committees ◽

Overall Response ◽

Risk Patients ◽

Grade 3

Abstract Abstract 2705 INTRODUCTION: This study aimed to determine the safety and maximum tolerated dose of the combination of everolimus and rituximab, or everolimus, bortezomib, and rituximab in relapsed and/or relapsed/refractory Waldenstrom Macroglobulinemia. This trial was based on our preclinical studies that demonstrated synergistic activity of everolimus and bortezomib with rituximab in WM cell lines, and based on our favorable clinical experience with everolimus as single agent in the treatment of WM. METHODS: Eligibility criteria include: 1) patients with relapsed or relapsed/refractory WM with any number of prior lines of therapy, including everolimus and bortezomib 2) not completely refractory to rituximab 3) measurable disease by monoclonal IgM protein in the serum and lymphoplasmacytic cells in the bone marrow, 4) Not receiving chemotherapy > 3 weeks, or biological/novel therapy for WM > 2 weeks. A cycle is 28 days and a total of 6 cycles are given, followed by everolimus maintenance for 2 years. Two stages with a total of four dose levels were planned. In stage A, patients received everolimus at the recommended dose orally daily for 28 days and rituximab at the recommended dose IV on days 1, 8, 15, and 22 every 28 days at cycle 1 and 4 only. In stage B, patients received everolimus at the recommended dose orally daily for 28 days, bortezomib at the recommended dose IV on days 1, 8, 15 every 28 days, and rituximab at the recommended dose IV on days 1, 8, 15, and 22 every 28 days at cycle 1 and 4 only. Patients were assessed for response after every cycle. Subjects who had a response continued on therapy for a total of 6 cycles, and then continued on to maintenance therapy with everolimus alone until progression (or for a maximum of 24 months). Because of the potential of an IgM flare after rituximab, patients who showed an increase in IgM after rituximab in the first 3 months were not deemed as having progressive disease unless they showed evidence of clinical progression. To examine the in vivo effect of everolimus, bortezomib, and rituximab, peripheral blood samples were obtained from patients on days 1, 8, 15, and 22 at cycle 1; and on day 1 only at all subsequent cycles. RESULTS: Twenty-three patients were enrolled in this phase I clinical trial from April 2009 to July 2011. The median age is 61 (range, 52–73) yrs and the median lines of prior therapy is 2 (range, 1–8) with all patients receiving prior rituximab and 12 (52%) receiving prior bortezomib. The median number of cycles on therapy was 3.5 (range, 0–15). Overall, this combination therapy is very well tolerated. Grade 4 toxicities included: neutropenia (8.7%), leukopenia (4.3%), thrombocytopenia (17.4%), lymphopenia (4.3%) and hypertriglyceridemia (4.3%). Grade 3 toxicities included: neutropenia (21.7%), leukopenia (26.1%), anemia (13%), lymphopenia (17.4%), pneumonitis (4.3%), SGPT (4.3%), neuropathy (4.3%), Herpes zoster reactivation (4.3%), hyperglycemia (4.3%) and hypernatremia (4.3%). 1 patient discontinued therapy due to grade 3 anemia. Nineteen patients are currently evaluable for response, including 1 (5%) very good partial response (VGPR) and 9 (47%) minimal response (MR), for an overall response rate including MR of 10/19 (53%) in this relapsed/refractory population. Furthermore, overall response including MR in stage A (everolimus/rituximab) was 2/6 (33%) and 8/13 (62%) in stage B (everolimus/bortezomib/rituximab). Additionally, 9 (39%) patients achieved stable disease, and 4 (17%) are early on therapy and not been yet assessed. CONCLUSIONS: The combination of everolimus, bortezomib, and rituximab is generally well tolerated, and importantly no grade 3/4 neuropathy was seen. Moreover, no dose limiting toxicities were observed even at the maximum dose evaluated. The responses observed to date in this relapsed/refractory population are encouraging. Based on the safety of this phase I study, the phase II study of two arms, everolimus/rituximab for low risk patients and everolimus/bortezomib/rituximab for intermediate and high risk patients is underway. Disclosures: Ghobrial: Bristol-Myers Squibb: Research Funding; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Bortezomib and everolimus in WM. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; 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; Acetylon: Membership on an entity's Board of Directors or advisory committees. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Treon:Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

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NVP-BEZ235, A Dual Inhibitor of Phosphoinositol-3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), Is a Potent Inhibitor of Lymphoma Cell Growth and Survival

Blood ◽

10.1182/blood.v118.21.4965.4965 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4965-4965 ◽

Cited By ~ 1

Author(s):

Daniela Buglio ◽

Manuela Lemoine ◽

Sattva S. Neelapu ◽

Francisco Vega ◽

Donald Berry ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Lymphoma Cell ◽

Parp Cleavage ◽

Lymphoma Cells ◽

Dual Inhibitor ◽

Growth And Survival ◽

Stat Pathway ◽

Cell Growth And Survival

Abstract Abstract 4965 The Phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR pathway is frequently deregulated in Hodgkin (HL) and non-Hodgkin lymphoma (NHL), and has been linked with tumor cell growth and survival. Although several proteins/enzymes in this pathway can be targeted by a variety of small molecules in vitro and in vivo, it remains unclear which protein target is the ideal for clinical testing. Previous studies demonstrated that the clinical activity of mTOR inhibitors may be attenuated by a negative feedback loop that involves activation of AKT, suggesting that a dual inhibition of AKT and mTOR activation may produce a better therapeutic outcome. To test this hypothesis, we evaluated the in vitro activity of NVP-BEZ235, a dual inhibitor of PI3K and mTOR, in a panel of 13 HL and NHL cell lines. NVP-BEZ235 inhibited cell growth and induced apoptosis in lymphoma cell lines in a time and dose dependent manner. After 48 hours of incubation, the IC50 ranged between 50 and 100 nM, and it was equally effective in ABC and GCB-derived DLBCL cell lines. NVP-BEZ235 induced cell death was primarily due to induction of apoptosis, as evident by the annexin-V and PI dual staining method, and the induction of caspase 3 and PARP cleavage. NVP-BEZ235 effectively inhibited the activation of the PI3K pathway at several steps, including decreasing the phosphorylation level of p-Akt (Ser473), p-Akt (Thr308), p-mTOR, p-4-EBPI and pP70S6K. Because lymphoma cells frequently depend on multiple activated signaling pathways to promote their survival, including the JAK/STAT pathway, we investigated the potential synergy between PI3K and JAK/STAT pathway inhibitors. Lymphoma cells were variably sensitive to the JAK1/2 inhibitor INCB16562 in vitro. Submaximal concentrations of NVP-BEZ235 demonstrated a synergistic activity with INCB16562. Collectively, our data show that the PI3K/mTOR inhibitor NVP-BEZ235 is highly effective against a wide range of lymphoma cell lines, and warrants evaluating it alone and in combination with JAK/STAT inhibitors in phase I/II clinical trials in patients with relapsed lymphoma. Disclosures: No relevant conflicts of interest to declare.

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