ALBERT

Description: Multiple myeloma (MM) remains incurable for the vast majority of patients due to emergence of drug resistant clones and mutations inducing drug resistant relapses. This is despite the fact that new therapies have greatly improved progression-free and overall survival for patients with standard risk myeloma. We recently found that the transcriptional repressor GFI1 is increased in bone marrow stromal cells of MM patients (MM-BMSC) where it causes prolonged suppression of osteoblast differentiation, and in CD138+ cells from MM patients, where GFI1 levels significantly correlate with disease progression. We also found that GFI1 overexpression (o/e) enhances MM cell growth and partially confers resistance to proteasome inhibitors in vitro as well as enhances tumor growth and osteoclastogenesis in vivo. Although the mechanisms responsible for these GFI1 effects in p53wt MM cells were p53-dependent, we found that GFI1 is also essential for MM cell survival regardless of their p53 status. The p53-independent mechanisms responsible for Gfi1 effects on MM cells growth and survival of are unknown. Sphingolipids are bioactive lipids that can control MM cell growth and survival. The balance between the levels of Sphingosine-1-phosphate (S1P) and its metabolic precursors ceramide (Cer) and sphingosine (SPH) form a rheostat that determines whether a cell proliferates or dies. We hypothesized that GFI1 represses SGPP1, the enzyme responsible for degrading S1P via salvage and recycling of sphingosine into long-chain ceramides. This repression changes the intracellular sphingolipid profile (Cer/S1P/SPH ratio) to maintain c-Myc upregulation in a protein phosphatase 2 (PP2A)-dependent manner, thus promoting growth and survival of MM cells. To test this hypothesis we measured S1P, SPH and Cer levels by mass spectrometry (LC-MS/MS). LC-MS/MS evaluation showed that bone marrow plasma of MM patients has significant higher levels of S1P when compared to normal donors. Moreover, intracellular S1P levels of MM.1S GFI1 o/e cells were also significantly higher as compared to those of MM.1S empty vector controls. Knock-down (KD) of Gfi1 in MM.1S cells strikingly increased SGPP1 and decreased SphK1 (the enzyme which catalyzes S1P production) mRNA levels, while GFI1 o/e cells had the opposite effect. We found that CD138+ cells isolated from MM patients expressed elevated levels of SphK1 mRNA compared to MGUS patients, and that SphK1 protein levels directly correlate with GFI1 levels in MM patient CD138+ cells and cell lines (r= 0.527). We also detected an indirect correlation (r= -0.961) between GFI1 and SGPP1 mRNA levels in five different MM cell lines. These results indicate a GFI1-dependent imbalance of the enzymes regulating S1P production. Further, KD GFI1 and SphK1 inhibition (5 μM SK1I) had a profound inhibitory effect on c-Myc protein levels and induced caspase 3 activation as detected by Western blotting, while GFI1 o/e cells had significant higher levels of c-Myc and were more resistant to SK1I treatment. Exogenous ceramide (10 μM Cer 16:0) treatment or SphK1 inhibition (5 μM SK1I), both treatments known to trigger intracellular ceramide production, significantly inhibited MM cell viability (measured by AlamarBlue), regardless of their p53 status (MM1.S p53 +/+ and KMS-11 p53 -/-). This inhibition of MM viability was GFI1-dependent, as GFI1 o/e cells were significantly more resistant to ceramide-induced cell death, which was PP2A dependent, as PP2A inhibition with okadaic acid (OA) restored it. MM.1S cells with KD of GFI1 exhibited significantly higher PP2A activity then control cells, supporting our observation that c-Myc modulation by GFI1 is PP2A-dependent. c-Myc protein levels were significantly decreased in MM.1S control cells treated with ceramide and rescued by OA pre-treatment; thus mimicking the effects of changing GFI1 levels and I2PP2A (the PP2A endogenous inhibitor) and confirms that PP2A mediates the effects of GFI1 on c-Myc. Taken together, our results show that GFI1 acts as a key regulator of MM growth and survival, at least partially through modulation of SGPP1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 3146 Background: High dose chemotherapy and stem cell transplantation (HDSCT) has been a standard of care for younger patients with adequate organ function since the early 1980s, due to its superiority over standard chemotherapy in prolonging disease-free and overall survival. Immunomodulatory agents, including thalidomide and lenalidomide, have significant single-agent activity and an additive effect when combined with melphalan. Preclinical data suggest an increased DNA damage and an anti-angiogenic effect with the combination. Additive clinical benefits were also observed when the 2 agents were used in combination in non-transplant settings. The antimyeloma effects of both agents are dose-dependent with myelosuppression being the dose limiting toxicity (DLT). This toxicity can be attenuated with stem cell rescue. Purpose: We conducted a phase I/II trial designed to evaluate the safety and efficacy of combining lenalidomide with high-dose melphalan as conditioning for autologous transplant for myeloma. The phase I portion of the study is complete and the results are reported in this abstract. Experimental Design: The enrolled patients included any patients with myeloma, regardless of the status of the disease, undergoing high dose melphalan for autologous stem cell transplantation. The melphalan dose was fixed (200 mg/m2) while the doses of lenalidomode were escalated from 50, 75, 100, and 150 mg/m2 administered orally days -7 to +2 of the transplantation. Dose escalation was based upon a 3+3 phase I design. DLTs were defined as grade ≥4, both hematologic and non-hematologic, occurring between days -7 to -2 which prevents subjects from undergoing stem cell transplantation, or grade 3 or 4 non-hematologic toxicity occurring after day -2 that does not resolve to a grade 2 or less by day +30 after transplantation, or delayed engraftment. The response was assessed at day +100 post transplantation using the International Myeloma Working Group criteria. Results: 13 patients participated in the phase I portion of the study from September 2010 to May 2012. Patients ages ranged from 42– 72 years (median 63 years). Seven patients were undergoing their first autologous transplant with 2 patients having had 2 lines of previous therapy and 5 having one line of therapy. Six patients were undergoing their second transplantationas salvage for control of progressive disease and all had more than 3 lines of prior therapy. At baseline, 5 patients had progressive disease, 1 had SD, 3 had PR, 3 had VGPR and 1 had CR as responses to their most recent lines of therapy. The median time for ANC and platelet engraftment was 10 days and no delayed engraftment was observed. Toxicities and posttransplant hematopoietic recovery rates were similar to historical data observed with single agent high dose melphalan. The most common grade ≥ 3 adverse events were myelosuppression, neutropenic fever and electrolyte abnormalities, all of which are commonly observed with single agent high dose melphalan. Adverse events related to the study drugs were electrolyte abnormalities (hypokalemia, hyperkalemia and hypocalcemia), gastrointestinal side effects and rash, all of which were grade 1 to 2 and manageable without a delay or discontinuation of the study drugs. One patient died from disease progression prior to scheduled disease evaluation. One patient has not yet reached day +100 post transplant. Therefore, responses were evaluable for 11 patients. Three patients achieved stringent CR (27%), 3 CR (27%), 2 VGPR (18%), 3 PR (27%) and one had progressive disease (9%). The overall response rate was 91%, with 72% achieving VGPR or better. All patients had adequate count recovery and were able to initiate lenalidomide maintenance treatment by day +100 to +110 post transplantation. Conclusions: The use of high dose lenalidomide in conjunction with high-dose melphalan is well tolerated, with preliminary data suggesting that the combination is highly efficacious. DLT was not observed; therefore the recommended phase II dose is 150 mg of lenalidomide orally on days -7 to +2 in combination with melphalan 200 mg/m2. The phase II portion of this trial is ongoing in patients undergoing first HDSCT. Complete response at 3 months post transplantation is the primary end point. Disclosures: Off Label Use: Lenalidomide in myeloma transplant. Abonour:Celgene: Honoraria, Speakers Bureau; Millenium: Honoraria, Speakers Bureau.

Description: Multiple myeloma (MM) is an incurable malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM) and by multiple lytic lesions throughout the skeleton. We previously reported that CD166 is a functional molecule on normal hematopoietic stem cells that plays a critical role in HSC homing to the BM and engraftment (Blood. 2014; 124(4):519-29.), suggesting that CD166 is involved in trafficking and lodgment of HSC. CD166, which is a member of the immunoglobulin superfamily capable of mediating both homophilic and heterophilic (CD6) interactions, has been shown to enhance metastasis and invasion in several tumors including breast cancer and melanoma. However, whether CD166 is involved in MM and whether it plays a role in MM progression or the bone lytic disease has not been addressed. H929-GFP MM cells were injected intravenously into NSG mice and GFP cells were recovered from the BM 14hr later. While only 29.9%±1.4% of total H929-GFP cells were CD166+, the frequency of CD166+ cells contained in BM-homed H929-GFP cells was significantly higher (80.0%±2.5%, n=9, p

Description: Background: The nuclear factor kappa B (NFkB) signaling pathway is constitutively activated and central to the pathogenesis of multiple myeloma (MM). We previously published the antitumor activity of the NFkB inhibitor parthenolide in MM. Dimethylaminoparthenolide (DMAPT) is a water-soluble analog of parthenolide. The improved bioavailability of DMAPT makes its appealing for clinical development. Objectives: To validate the effects of DMAPT both in vitro and in vivo MM models and to develop a rational strategy for a combination of DMAPT with other anti-MM agents. Methods: The in vitro cytotoxicity of DMAPT was tested in human MM cell lines and primary samples both alone and in combination with bortezomib and lenalidomide. The in vivo activity of DMAPT was tested in a model of NOD/Scid mice intravenously transplanted with GFP-tagged NCI-H929 MM cells. Results: Based on MTT assays on MM cell lines and primary MM cells, the IC50 for proliferation inhibition of DMAPT was similar to that of parthenolide (1–3 μM). When used in combination with bortezomib, we observed synergy (combinatorial index 〈 1.0) at higher doses of DMPT and bortezomib (DMAPT 2.5–5 μM and bortezomib 5–12.5 nM), while an antagonistic effect was observed at lower doses. Similarly, when combined with lenalidomide, synergy was observed at DMAPT doses 1.6–6.4 μM and lenalidomide doses 0.5–2 μM, while an antagonistic effect was observed at lower doses. The in vivo cytotoxicity of DMAPT was explored in a model of NOD/Scid mice intravenously transplanted with GFP-tagged NCI-H929 cells. At 3 weeks post-transplantation, all animals had measurable serum human kappa light chain levels, as measured by ELISA assays. Animals were randomized to receive either 50 mg/kg/day DMAPT (n=12), 100 mg/kg/day DMAPT (n=12) or vehicle (n=10) via oral gavage for a duration of 6 weeks or until criteria for euthanasia were met. Weight loss of over 15% and hind leg paralysis were criteria for euthanasia. Toxicities in both treatment groups were minimal. Weight loss correlated with serum human kappa light chain levels and, hence, represented disease progression. At the end of treatment, there were 2 animals alive in the control group, while 5 were alive in each of the treated groups. Using Kaplan-Meier curves and log-rank analysis, the mean overall survival (OS) was 56.3 days in the control cohort versus 61.0 days and 62.4 days in groups treated with 50 and 100 mg/kg/day DMAPT, respectively. There was no statistically significant difference in the mean OS of mice treated with the two doses of DMAPT (p=0.07). Prior to treatment initiation, the serum levels of human kappa light chains were similar in all groups (ANOVA test, p= 0.38). At 2 weeks and 4 weeks post treatment (Week 5 and week 7, respectively; See table), the levels of human kappa light chains were significantly lower in the DMAPT treated groups (ANOVA test p = 0.017 and 0.0061, respectively). Week 3 Week 5 Week 7 Control 10.46 ± 5.79 531.31 ± 238 6272.1 ± 3211 DMAPT 50 mg/kg/day 6.94 ± 4.48 264.03 ± 198 2928.8 ± 2131 DMAPT 100 mg/kg/day 7.99 ± 7.21 334.15 ± 198 3374.9 ± 1835 ANOVA (P value) 0.38 0.017 0.0061 Conclusions: DMAPT demonstrated excellent efficacy in preclinical models of MM and supports a rationale for its clinical development in MM patients with relapsed MM either alone or in combination with bortezomib or lenalidomide.

Description: Although monocytic zinc finger protein (MOZ/MYST3) maintains normal hematopoietic stem cells, its fusion to the coactivator CREB-binding protein (CBP/CREBBP) induces acute myeloid leukemia (AML). Since leukemic stem cells in AML often exhibit excessive signal-dependent activity of the transcription factor NFκB, we hypothesized that cooperation between NFκB and MOZ-CBP represents an alternative mechanism for enhancing NFκB transcriptional activity. In reporter assays, MOZ and CBP separately induce transcription from the NFκB-dependent interleukin-8 promoter; however, these two proteins together markedly activate its expression. Although MOZ has less potent transcriptional activity than MOZ-CBP, both proteins cooperate with steroid receptor coactivator-1 (SRC1/NCOA1) to activate transcription. Since cooperation between MOZ-CBP and SRC1 is strongly reminiscent of the interaction between MOZ-TIF2/SRC2 and CBP required to induce AML (Deguchi et al, 2003), these findings suggest that MYST family fusion proteins may assemble a conserved leukemogenic transcriptional complex composed of a MYST protein (MOZ, MORF), molecular integrator (CBP, p300), and nuclear receptor coactivator (SRC1, TIF2/SRC2). MOZ also induces multiple NFκB-dependent viral promoters. Importantly, MOZ associates in a protein complex with the DNA-binding p65/RELA subunit of NFκB in vivo and interacts directly with p65 in vitro. Whereas deletion of the leukemia-associated protein (LAP/PHD) or MYST domains decreases the transcriptional activity of MOZ, deletion of either the acidic domain or C-terminal domain completely abrogates its activity. Since the C-terminal domain is absent from MOZ-CBP, these results indicate that the potent transcriptional activity of MOZ-CBP represents a gain-of-function property derived from the retained portion of CBP. Collectively, these studies not only demonstrate that MOZ and MOZ-CBP cooperate with NFκB to enhance expression of NFκB-dependent promoters but also suggest that aberrant interaction between MOZ-CBP and NFκB may play an important role in the pathogenesis of certain acute myeloid leukemias.

Description: Multiple myeloma (MM) bone disease (MMBD) is characterized by activation of osteoclasts and suppression of osteoblastic differentiation, with these changes in the bone microenvironment supporting MM cell growth and drug resistance. These complex interactions between MM cells and bone cells are incompletely understood. Current bone targeted therapy with bisphosphonates or Denosumab only blocks bone resorption but has no effect on osteoblast activity and only modest effects on MM growth. Therefore, new MMBD treatments are needed. Semaphorin-4D (Sema4D; CD100), is made by osteoclasts and inhibits osteoblasts by binding to the Plexin B receptor. Breast cancers also express Sema4d, and silencing sema4D in MDA-MB-231 breast cancer cells suppresses bone metastasis (Yang Y et al, PLoS One 2016). Since breast cancers and MM both cause osteolytic bone destruction and soluble Sema4D and Plexin B levels are increased in sera of MM patients (Terpos et al, 2012), we tested if sema4D contributed to MMBD. qPCR analysis of human MM cell lines and primary CD138+ cells showed MM cells express high levels of sema4D mRNA, comparing to the MDA-MB-231 breast cancer cells. Analysis of previously reported gene expression array data confirmed that MM cells express sema4D at a higher level compared to bone marrow plasma cells of MGUS and healthy donors (GenomicScape.com; Zhan F et al, Blood 2007; Mattiolo M et al, Oncogene, 2005). These results plus those of Terpos et al suggest that MM cells commonly express Sema4D. We next asked if the bone microenvironment increases MM expression of Sema4D. We co-cultured human MM cell lines RPMI8226 and JJN3 with mouse bones. Species -specific changes in tumor and bone were evaluated by quantitative RT-PCR. MM cells engrafted onto mouse bones, increasing markers of osteolysis similar to those seen in MM bone disease. After a week of co-culture, Sema4D expression was increased in MM cells (mean ±SD; 4.2±0.4; p=0.023), compared to MM cells grown alone. In addition, bones co-cultured with MM cells expressed higher Sema4D mRNA than bones alone (mean ±SD; 3.6±0.21; p=0.03). While co-culture increased both MM and bone Sema4D, markers of osteoblast activity, Col1a1, alkaline phosphatase and osteocalcin were suppressed. Preliminary experiments suggest that osteocytes are a major source of Sema4D expression in bone, in addition to active osteoclasts, which are much rarer cells than osteocytes. The induction of Sema4D in bone was only partially inhibited by 100nM zoledronic acid to inhibit osteoclast activity. Since osteocytes can physically interact with MM cells in vivo (Delgado Calle, Cancer Res 2016), we then tested the effect of MM cells on osteocyte sema4D expression in co-cultures of RPMI 8226 and JJN3 MM cells with MOL-Y4 osteocytic cells, separated by transwells. Both MM cell lines increased the Sema4D mRNA content of MLO-Y4 cells (mean ±SD; 3.1±0.4; p=0.036), suggesting that myeloma-secreted factors regulate osteocyte Sema4D expression. Since Sema4D is a potent osteoblast inhibitor, our data suggest that osteocyte -derived Sema4D may be a major contributor to MMBD, and that neutralization of Sema4D activity should improve the suppressed bone formation in MM. Disclosures Roodman: Amgen: Consultancy.

Description: Abstract 1966 Poster Board I-989 The Effect of Targeting the Antiapoptotic Protein c-FLIP in Multiple Myeloma Background: Multiple myeloma (MM) is an incurable blood cancer. Treatments which target key proteins that play a role in drug resistance should improve treatment outcomes. The cellular FLICE-inhibitory protein (c-FLIP) is an antiapoptotic protein which confers resistance to death receptor-mediated apoptosis. Further, c-FLIP overexpression has been identified in various cancers, and in MM specifically, gene expression profiling has demonstrated that c-FLIP is overexpressed in a patient's MM cells compared to the normal plasma cells of an identical twin. However, the precise clinical significance of c-FLIP overexpression and its potential as a therapeutic target in MM have not been established. Objectives: To determine the potential of c-FLIP as a therapeutic target by exploring the in vitro and in vivo effects of c-FLIP knockdown in MM cells, using a doxycycline-inducible lentiviral vector containing c-FLIP shRNA. Results: Doxycycline treatment in vitro induced c-FLIP shRNA transcription and reduced c-FLIP levels more than 80% in H929 cells. Correspondingly, we observed a greater than 95 % reduction in the growth of H929 cells, compared to the cell line containing an empty vector control. In addition, immunoblots of MM cell lysates showed an accumulation of the cleaved products of caspases 8 and 3, suggesting that c-FLIP knockdown sufficiently induced caspase activation and apoptosis in MM cells. To explore the cytotoxicity of c-FLIP knockdown in MM cells in vivo, we used two different mouse models. In the first model, MM cells containing the doxycycline inducible, c-FLIP shRNA containing, lentiviral vector, were transplanted subcutaneously into NOD/Scid mice. Two weeks post transplantation, when accurate tumor measurements could be made, the mice were divided into two groups, of approximately equal tumor volumes, and one group was given doxycycline treated water. A significant reduction in tumor size was observed in doxycycline treated mice compared to the controls (Figure 1). In a second mouse model, NOD/Scid mice were transplanted with MM cells by tail vein injection and divided into two groups. The first group (“early dox”; n=10) was given doxycycline treated water 4 hours post transplantation, while the second group (n=20) was given regular water until they developed tumors which could be determined by measuring human IgA kappa levels in the mouse serum. Within this group, mice were randomized base on the IgA levels to receive doxycycline treatment (“late dox”; n=6) or regular water (n=6). ELISA measurements of human IgA kappa light chain from treated mice are shown in Figure 2. The “early dox” group had barely detectable IgA kappa levels while the “late dox” group had significantly lower levels than the mice that never received any doxycycline. Summary: An inducible c-FLIP shRNA system was created in order to observe the effects of c-FLIP knockdown on the viability of MM cells both in vitro and in vivo. A nearly complete c-FLIP knockdown could be generated in vitro which had a dramatic effect on the viability of the MM cells, and this translated to a significant reduction in tumor volumes when these cells were later transplanted into mice, both subcutaneously and intravenously, and then treated with doxycycline. This work provides evidence that targeting c-FLIP in MM, either directly or via gene regulation, holds significant promise. Disclosures: Abonour: Celgene: Honoraria; Millennium: Honoraria.

Description: ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTK). We investigated the single agent activity of ENMD-2076 against MM cells in vitro and in vivo, and in combination with lenalidomide. ENMD-2076 free base showed significant cytotoxicity against MM cells with a mean LC50 of 3.84±0.86 μM at 48 hours in vitro. Cytotoxicity was associated with cleavage of caspase 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. ENMD-2076 free base inhibited c-kit, FGFR-1, 3 and VEGFR1 and subsequently inhibition of downstream targets phosphorylated (p)-BAD, p-Foxo1a and p-GSK-3β was observed at 6 hours. NOD/SCID mice implanted with H929 human plasmacytoma xenografts and treated for 30 days with 50, 100, 200mg/kg/d ENMD-2076 showed a dose-dependent inhibition of tumor growth (Figure 1), with minimal toxicity as assessed by the stable weight of treated animals. Immunohistochemical staining of tumors from sacrificed animals showed significant reduction in Ki67 at all dose levels of treatment compared to control tumors. An increase in cleaved caspase-3 was observed on Western blot from the lysates of H929 tumors obtained from treated animals. ENMD-2076 free base also showed synergistic cytotoxic activity when combined with lenalidomide against H929, MM1.R and MM1.S cells as assessed by MTT assay and Annexin-V/PI staining. Using the Chou-Talalay method, the combination indices (CI) were 〈 1 for all three cell lines across a range of concentrations of ENMD-2076 free base (0.25–1.0 μM) plus lenalidomide (2.5–10 μM) indicating synergistic activity (CI=0.362 H929; CI=0.315 MM1.R; CI=0.415 MM1.S). Our results provide rationale for the investigation of ENMD-2076 alone and in combination with lenalidomide in patients with multiple myeloma. Figure Figure