Anderson, Kenneth C.

Abstract In an effort to identify novel genetic abnormalities in multiple myeloma (MM) we analyzed an initial set, as part of the Multiple Myeloma Research Consortium (MMRC) Genomics Initiative, of 94 MM patient samples on high-resolution array-based comparative genomic hybridization (aCGH) and gene expression profiling (GEP) technologies. The MMRC Genomics Initiative is a three-year research program spanning a full-spectrum of genomic technologies with the goal of analyzing 250 MM patient samples by 2009. Raw data from the Initiative are released in near real time through the MMRC Multiple Myeloma Genomics Portal. This initial aCGH dataset from the Agilent 244K platform represents the highest resolution gene copy number analysis with corresponding GEP released to date in MM. It has confirmed the cytogenetic definitions of MM with the data set being split between patients with hyperdiploid and non-hyperdiploid MM. Furthermore, the 244K platform has helped to refine minimal regions of change present on 1p, 1q, 6q, 8p, 13q, 14q, and 16q. A comprehensive screen of the dataset for genes residing in regions of homozygous deletion identified 105 genes. The most commonly affected gene was CDKN2C(p18) (8.3%) while 12 other regions were recurrently targeted. Several of these regions were recently implicated in the pathogenesis of MM but the large majority represents novel observations. The aCGH and GEP data were integrated using two different programs developed by the core institutions (The Broad Institute of Harvard & MIT and Translational Genomics Research Institute). First, Genomic Identification of Significant Targets in Cancer (GISTIC), which identifies highly significant regions of change and a limited set of candidate genes within these regions, was used to identify 285 genes associated with DNA content alterations. Second, Breakpoint Expression Correlation (BEC), which identifies dysregulated genes on either side of a copy number alteration, was used to identify 194 genes with expression changes associated with a breakpoint. This latter gene list includes all known IgH translocation target genes identified to date in myeloma. Overlapping the gene lists generated by GISTIC, BEC, and the homozygous deletion screen identified a number of genes previously implicated in MM such as CDKN2C(p18), CYLD, BIRC2/BIRC3, and two novel genes, FAM46C and RNF6. Ongoing data integration with RNAi and high content sequencing projects of the current Initiative holds great promise in furthering our understanding of MM. The MMRC Genomics Initiative and resulting efforts from the myeloma research community will increase our understanding of MM at the molecular level, help to identify new targets, and ultimately lead to the development of better, more effective therapies for myeloma.

Abstract PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Abstract The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative is a three-year program to analyze tumor tissue from hundreds of multiple myeloma (MM) patients via gene expression profiling (GEP), comparative genomic hybridization (aCGH), and exon re-sequencing. In addition, RNAi knockdown of selected genes in MM tumor cell lines is being evaluated to identify potential new targets. All genomic data generated is scheduled for placement in an open-access Multiple Myeloma Genomics Portal pre-publication and in near real-time (www.broad.mit.edu/mmgp). Additionally, samples are also destined for drug validation and correlative science on clinical protocols as this study moves forward. This comprehensive project is spearheaded by the MMRC and conducted via collaboration with the Eli and Edythe L. Broad Institute of MIT and Harvard, the Translational Genomics Research Institute (TGen), Mayo Clinic Arizona, and The Dana-Farber Cancer Center. The study is supported by the collection from member institutions of the MMRC of bone marrow aspirates and matched peripheral blood samples from over 1000 patients. Specific genomic technologies that are currently being employed across this sample set include GEP using Affymetrix Human Genome U133A 2.0 Plus Arrays, and, in parallel, efforts to identify regions of genomic gain and loss are using Agilent Human Genome CGH arrays. In contrast to other large-scale genomic projects based on exon-sequencing of targeted gene sets, this project will be the first to perform genome-scale single molecule sequencing (SMS) of DNA from patient specimens. Results will be targeted against candidate classes of genes (e.g. kinases, phosphatases, known oncogenes and tumor suppressors), and genes from GEP or within candidate regions of copy gain or loss identified by the aCGH experiments. Mutations will be further validated in an independent set of patient specimens. Finally we will attempt to identify points of vulnerability of MM through systematic loss-of-function screens in myeloma cell lines using high-throughput RNA interference (using both shRNA and siRNA platforms). Importantly, data generated from this genomics initiative will ultimately be made public pre-publication through the established MMRC Multiple Myeloma Genomics Portal. Data from all aspects of this project (sample collection and analyte isolation, GEP, aCGH, SMS, RNAi and bioinformatics) will be described in this presentation. The power of this study is the comprehensive collection of gene expression, CGH, and genome sequencing on a single reference set of clinically annotated samples. The addition of RNAi screens makes this a very important and unique data resource, which we hope will help expedite the discovery of novel targeted agents for MM scientific community.

Abstract The importance of bone marrow angiogenesis in multiple myeloma (MM) provided the initial rationale for using thalidomide in this disease, given its anti-angiogenic properties. Thalidomide (α-N-[phthalimido] glutarimide, C 13 H 10 N 2 O 4), a glutamic acid derivative, has proved useful in the treatment of both newly diagnosed and relapsed/refractory MM, however, its potential for teratogenicity lays a huge burden on its use as a therapeutic agent. Ongoing efforts have been directed towards development of analogues, which retain anti-MM activity without teratogenic effects. A product of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5′-OH-thalidomide, has been shown to be partly responsible for the anti-angiogenic effect of thalidomide. Based on the structure of this metabolite several N-substituted and tetraflourinated thalidomide analogues have been synthesized as potential anti-angiogenic agents and have shown pre-clinical activity in prostate cancer. In this study, we demonstrate potent anti-MM activity and delineate potential mechanisms of two such compounds, CPS11 (an N-substituted thalidomide) and CPS49 (a tetraflourinated thalidomide). Both CPS11 and CPS49 showed inhibition of proliferation of several MM cell lines as well as primary tumor cells from relapsed/refractory MM patients. These drugs had significant activity in myeloma cell lines resistant to conventional chemotherapy agents (dexamethasone, adriamycin, mitoxantrone and melphalan). Both the drugs were able to overcome the protective effects of growth factors like IL-6, IGF-1, and VEGF, and co-culture with bone marrow stromal cells (BMSCs), demonstrating their activity against myeloma cells in conditions simulating the natural microenvironment. In most of the cytotoxicity experiments, CPS49 was more potent compared to CPS11. An additive cytotoxic effect was noted when these agents were combined with dexamethasone and melphalan. Apoptosis was noted in MM1.S cells as evidenced by annexin and PI staining and a time- and dose-dependent increase in cleavage of poly ADP-ribosepolymerase (PARP) and caspase-8. Additionally, Z-VAD-FMK partially blocked these effects. Importantly, apoptosis triggered by these drugs was associated with down-regulation of anti-apoptotic proteins like Mcl1 and XIAP. In matrigel based angiogenesis assays both drugs demonstrated anti-angiogenic activity, CPS49 being more potent than CPS11. To further delineate their molecular mechanism of actions, we evaluated the effect of these drugs on various intracellular signaling pathways known to be important in myeloma. Both the drugs were able inhibit the PI3K/Akt and JAK/STAT pathways in MM1.S myeloma cells line. These encouraging in vitro data will provide the platform for testing these analogues initially in animal models and subsequently in the clinic.

Abstract PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study did not respond. Lysophosphatidic acid (LPA) is a phospholipid which mediates tumor cell migration and invasion. Recent studies have shown that inhibition of LPAAT-β inhibits both Ras/Raf/Erk and PI3K/Akt signaling cascades. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-β inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (ie, dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 lymphoma cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor (DFF) 45 is triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. Here we show that PS-341 does not induce phosphorylation of SEK-1, JNK, and c-Jun in DHL-4 cells, suggesting that it does not trigger a stress response. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggers swelling and cell membrane destruction in DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-β inhibitor CT-32615 triggers necrosis even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.

TPS8057 Background: Multiple myeloma (MM) is the second most prevalent blood cancer, representing approximately 1% of all cancers. Although overall survival has improved in recent years due to new approved agents, the vast majority of MM patients (pts) ultimately stop responding to treatment. Moreover, about a quarter of MM pts characterized as “high risk” experience limited benefit from existing treatments. Seminal genomic sequencing research efforts, such as the MMRF CoMMpass study, have highlighted that a large number of MM cases harbor potentially actionable oncogenic molecular alterations and published reports on small numbers of cases suggest that Precision Medicine (PM) interventions clinically targeting such actionable drivers may benefit MM pts. These results suggest that PM approaches in MM are possible and should be further studied clinically. To that end, we have launched MyDRUG, a master protocol aimed at developing new myeloma regimens based on individual pt’s genomics. Methods: This study is a Phase I-II, multicenter master protocol in functional high risk MM patients (i.e patients having progressed from baseline treatment within 18 months without maintenance or 3 years on maintenance) with 1-3 prior lines of therapy. Patients are screened for actionable genomic alterations on the CLIA-grade MI-ONCOSEQ platform. Patients with actionable alterations are assigned to the appropriate targeted agent used in combination with a backbone regimen ixazomib, pomalidomide and dexamethasone (IPd), whereas patients without such alterations go on an immune arm (see Table). Inclusion criteria include measurable disease (as measured by M-protein and FLC) and acceptable hematologic and metabolic functions. A maximum of 12 evaluable pts will be accrued onto the phase I portion of each arm and an additional 21 evaluable phase II pts will be accrued onto each arm for a total of 27 evaluable pts at the MTD combination dose. The primary objective of the study is to evaluate ORR per IMWG consensus criteria. Secondary objectives are to assess adverse events (AEs), progression-free survival, and overall survival (OS). Experimental correlative aims include assessing molecular or clonal response, molecular and immune signatures of resistance/response, MRD. Clinical trial information: NCT03732703. [Table: see text]

8004 Background: The primary objective of this phase 3 trial was to demonstrate progression free survival (PFS) improvement of isatuximab (Isa), a novel anti-CD38 monoclonal antibody, combined with pomalidomide (P)/dexamethasone (d) versus (vs) Pd. Methods: Patients (pts) with RRMM who received ≥2 prior lines, including lenalidomide (len) and a proteasome inhibitor (PI), refractory to last therapy were enrolled. IsaPd arm received Isa 10 mg/kg IV weekly for first 4 weeks (wks), then every 2 wks. Both arms received approved schedules of pom and dex (4mg PO days 1-21; 40mg [20mg if >75 yrs] PO or IV weekly) every 28 days until progression or unacceptable toxicity. Results: 307 pts (154 IsaPd, 153 Pd) were randomized and analyzed (ITT). Patient characteristics were well balanced across arms. Median age: 67 (36-86) yrs; median prior lines of therapy: 3 (2-11); estimated GFR: <60ml/min in 33.9% pts; 92.5% refractory to len, 75.9% to PI; and 19.5% pts had high-risk cytogenetics. At median follow-up of 11.6 months (mos), median PFS was 11.5 mos IsaPd vs 6.5 mos Pd; HR 0.596 (95% CI 0.44-0.81), P=0.001. PFS benefit was consistent across all major subgroups. ORR (≥PR) was 60.4% IsaPd vs 35.3% Pd, P<0.0001. VGPR rate or better was 31.8% IsaPd vs 8.5% Pd, and MRD negativity (NGS, 10-5) was seen in 5.2% IsaPd pts vs 0% Pd. At analysis date, overall survival (OS) was immature (99 events) but a trend to OS improvement in IsaPd (vs Pd) was observed (HR 0.687; 95% CI 0.461-1.023). Median treatment duration was 41 wks IsaPd vs 24 wks Pd; median Isa infusion (inf.) duration was 3.3h at 1st inf. and 2.8h at subsequent inf. Grade ≥3 AEs were observed in 86.8% IsaPd vs 70.5% Pd; 7.2% IsaPd and 12.8% Pd pts discontinued due to AEs; 7.9% IsaPd and 9.4% Pd pts died due to AEs. Inf. reactions were reported in 38.2% (2.6% grade 3-4) IsaPd. Grade ≥3 infections were seen in 42.8% IsaPd and 30.2% Pd, grade ≥3 neutropenia in 84.9% (febrile 11.8%) IsaPd and 70.1% (febrile 2.0%) Pd. Conclusions: IsaPd significantly improved PFS and ORR vs Pd, with a manageable safety profile. IsaPd is an important new treatment option for the management of RRMM. Clinical trial information: NCT02990338.

8592 Background: Accrual to NCI clinical trials(CT)is often slower than planned at times mandating premature closure resulting in loss of valuable resources and delay of scientific progress. Methods: The NCI-MYSC AWG identified 10 potential BtA. SWOG, ECOG and Alliance investigators were queried and agreed that these barriers impede accrual (results stratified for academic and community sites). The MYSC AWG developed in collaboration with NCI and FDA strategies to overcome these barriers. Results: Strategies listed for the 3 most often cited BtA: 1. Reimbursement for CT related expenses:increase awareness of improved reimbursement for phase II CT; tailor reimbursement according to CT complexity; request funds from industry and other sources ( http://biqsfp.cancer.gov ) for qualifying ancillary CT components. 2. Spectrum of available treatment options influences CT participation: educate patients and providers about the significance of a new CT using social media, presentations at national meetings and by adding educational material to CT protocol; encourage opinion leaders and advocacy groups not to promote a new therapy as “standard” in the absence of phase III data. 3. Requirement of CT specific therapy at NCI designated sites only: “MYSC AWG Drug Administration Table” describes NCI/FDA approved rules for CT specific drug administration; CT protocol will outline which standard treatment components of a CT can be administered at any site as long as protocol specific guidelines are followed and conduct is supervised by enrolling investigator. Examples of additional strategies to overcome identified BtA: determine feasibility, indication and insurance coverage of CT specific tests during protocol development; discourage narrow eligibility criteria; avoid competing CT; allow up to 1 cycle of commercially available therapy prior to enrollment; CIRB support for phase II CT. Conclusions: The MYSC Accrual Working Group developed in collaboration with NCI and FDA strategies to overcome barriers to myeloma clinical trial accrual. These strategies may be applicable to NCI-sponsored clinical trials evaluating interventions in other diseases.

8043 Background: The mammalian target of rapamycin (mTOR) signal pathway controls cell proliferation and survival. The trial's goal was to determine the anti-tumor activity and safety of single-agent everolimus (TORC1 inhibitor) in patients with relapsed/refractory Waldenstrom macroglobulinemia (WM). Methods: Eligible patients had measurable disease (IgM monoclonal protein >1000 mg/dL with >10% marrow involvement or nodal masses >2 cm), a platelet count ≥75,000 x 106/L, a neutrophil count ≥1,000 x 106/L. Patients received everolimus 10 mg PO daily. Tumor response was assessed after cycles 2 and 6 and then every 3 cycles until progression. Results: 60 pts were treated. The median age was 64 years (range, 43-85). The median number of prior therapies was 3 (range, 1-11). All but two patients (97%) had received prior rituximab based therapy and 64% of patients had received prior alkylator based therapies. The overall response rate (complete response CR+ partial response PR+ minimal response MR) was 73% (95% CI: 60-84%), with a PR of 50% and 23% MR. The median time to progression (TTP), progression-free survival (PFS), and overall survival (OS) for the entire study population is 28 months (mos), (95% CI: 18-not reached (NR)), 22 mos (95% CI: 12 0-NR), and 55 mos (95% CI: 55-NR), respectively. The estimated PFS at 12 and 24 months is 62% (95%CI: 51-75%), and 48% (95%CI: 37-63%), respectively. The 30 patients who achieved a PR responded after a median of 2 months (range, 1-26) of treatment. The median duration of response (DR) for these patients has not yet been reached and 19 of these patients remain in response after a median follow up of 31 months (range, 3-54). All but two patients had a decrease in their serum IgM. Grade 3 or higher related toxicities were observed in 67% of patients. The most common were hematological toxicities with cytopenias. Pulmonary toxicity occurred in 5% of patients. Dose reductions due to toxicity occurred in 63% of patients. Conclusions: Everolimus has high single-agent activity with an overall response rate of 73% and manageable toxicity in patients with relapsed WM, and offers a potential new therapeutic strategy for this patient population.

17502 Background: CHIR-258 is an orally active small molecule receptor tyrosine kinase (RTK) inhibitor which has potent activity against multiple RTKs involved in tumor growth and angiogenesis (IC50 ≤13 nM for FGFR, VEGFR, PDGFR, C-KIT, and FLT3). Approximately 15% of newly diagnosed MM patients harbor a t(4;14) translocation, which results in ectopic expression of FGFR3 and MMSET and is associated with poor treatment outcome [0]and reduced survival. CHIR-258 inhibits proliferation and induces apoptosis in FGFR3-expressing MM tumor xenografts as well as FGFR3-expressing primary MM cells (Trudel et al, Blood 2005). CHIR-258 was synergistic with dexamethasone (dex) in vitro. Methods: CHIR-258 was administered once daily in a dose-escalating phase 1 study to patients with relapsed/refractory MM. Drug tolerability and safety, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed. Results: as of December 2005, 9 total pts have been treated (50, 100, and 200mg qd cohorts) [6M, 3F; median age: 58 (range: 44–68), median of 3 prior therapies(range:1–7)]. Prior tx: 9/9 pts-thalidomide; 8/9 -Velcade; 8/9-transplant; 8/9- had progressed through dex. 4 of 9 pts treated were FGFR3+ and 4 pts remain on study (3 are FGFR3+). No CR or PR have been observed; stable disease has been noted. CHIR-258 has been generally well tolerated, and most drug related AEs were CTC grade 1 or 2, including: headache, dysgeusia, fatigue and anorexia. No neuropathy. One DLT has been observed to date: neutropenia (200mg cohort). Five pts had dex added to CHIR-258 of which 3 are ongoing (all FGFR3+). FGFR3+ pts receiving dex and CHIR-258 have a greater decline in urine and serum paraproteins (pp) vs CHIR-258 alone. Plasma exposure and Cmax increased proportionally across the doses. Conclusions: CHIR-258 is a novel inhibitor of RTKs involved in MM growth and proliferation. FGFR3+ pts have a more marked reduction in pp than FGFR3- pts. CHIR-258 has generally been well tolerated and further accrual continues. [Table: see text]