Ness, Brian Van

Mutations in genes that code for RAS isozymes (N- and K-) are the most common (40-50%) somatic variations in multiple myeloma (MM). While ~46% of patients with newly diagnosed MM carry a RAS/Raf mutation, >64% of patients with refractory/relapsed MM carry these mutations. RAS activation can also occur from upstream signaling through receptor tyrosine kinases (RTK) or loss-of-function mutations in GTPase-activating proteins (GAPs), which are prevalent in MM. Although covalent inhibitors targeting KRAS alleles are in various stages of clinical development, multiple mechanisms of resistance have been reported. We discovered and developed a novel, highly potent, and selective pan-RAS inhibitor, ADT-007, capable of suppressing MAPK/AKT signaling by blocking GTP activation of RAS. Here, we show that ADT-007 is effective against MM, particularly those MM cell lines harboring KRAS and NRAS mutations that are representative of relapsed/refractory myeloma (RRMM). For these studies, we compiled a panel of over 50 human myeloma cell lines (HMCLs) with broad biological and genetic heterogeneity that represent innate drug-response/resistance (refractory disease), as well as 10 pairs of parental and clonally-derived proteasome inhibitor (PI)- and immunomodulatory drug (IMD)- resistant HMCLs as models of acquired resistance (relapse). Using CRISPR-cas-directed gene editing, we also have generated clonal NRAS and KRAS mutants of drug-sensitive myeloma lines that initially had two wild-type alleles. All cell lines have been authenticated using the GenePrint 24 System and tested for mycoplasma contamination. First, we performed predictive analysis using single-cell RNA sequencing (scRNAseq; 10X Genomics) on all MM cell lines and demonstrated the potential efficacy of ADT-007 based on the subclonal expression of the target genes/pathways. Using in vitro chemosensitivity assays, ADT-007 was highly potent and effective in killing chemo-resistant and RAS-mutant myeloma cells as a single agent with IC50 values ranging from 0.76 to 12 nM with synergistic benefits if combined with PIs and IMDs. Combination indices (CI) were found to be consistently below 0.9, indicating a synergistic drug interaction. (Drug synergy was determined by CalcuSyn software based on Chou-Talalay's CI method and the isobologram algorithm). Caspase 3/7 assay and flow cytometry (Annexin V-FITC + propidium iodide) confirmed apoptosis induction by ADT-007. Furthermore, the in vitro efficacy of ADT-007 was significantly better (nM vs. µM IC50) than cyclorasin B4-27, a bicyclic peptidyl that has been reported as a potent pan-RAS inhibitor against several other cancers, and RMC-6236, a multi-selective noncovalent RAS(on) inhibitor which is in clinical trial for patients with advanced solid tumors harboring G12X, G13X, and Q61X mutations. Pre-vs-post-treatment bulk-RNA sequencing followed by pathway analysis (using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis) showed that ADT-007 suppressed KRAS and NRAS in a dose-dependent manner. Immunoblotting analysis confirmed the dose-dependent reduction of protein markers implicated in cell survival and chemotherapeutic resistance in myeloma, including c-myc, Ki-67, NFkB, IRF4, IKZF1 (Ikaros) and IKZF3 (Aiolos). Finally, we performed pre-vs-post-treatment single-cell transcriptomics analysis, which revealed a distinct shift in clusters representing myeloma single-cell subpopulations following ADT-007 treatment. High-dimensional mass cytometry (cytometry by time of flight or CyTOF) analysis using heavy metal-conjugated proteins in RRMM patient bone marrow-derived primary myeloma cells is currently underway to confirm the cytotoxic effect of ADT-007 ex vivo. ADT-1004 is an orally bioavailable prodrug of ADT-007 designed to improve water solubility and metabolic stability. ADT-1004 has shown strong antitumor activity in highly aggressive and clinically relevant mouse models of pancreatic cancer at dosages, is well tolerated, and is being developed by the NCI Stepping Stones program. Next, we plan to evaluate the antitumor activity of ADT-1004 in mouse xenograft models of human myeloma using immunocompromised mice. Our research will lay the groundwork for future development efforts needed to advance ADT-1004 to clinical trials involving patients with RRMM.

Abstract Although multiple myeloma is clinically defined as the accumulation of clonal, malignant plasma cells in the bone marrow, the disease shows significant heterogeneity with regard to progression and therapeutic response. It is likely that germline genetic polymorphisms contribute significantly to an individual’s disease course and response. Bank On A CureTM (BOAC) is a collaborative project initiated with the International Myeloma Foundation (IMF) to develop a DNA bank of myeloma patient samples, through international cooperation. As the bank continues to expand, it provides a resource for the development of cooperative partnerships to assess specific clinical trials as well as conglomerate data. In this study, we have combined data from 3 phase III trials: ECOG trial E9486 (VBMCP + cyclophosphamide or interferon); SWOG trial 9321 (VAD induction followed by randomization to PBSC-supported high dose therapy versus standard dose therapy of VBMCP); and the MRC7 trial (comparing conventional chemotherapy [ABCM] with intravenous chemotherapy and high dose melphalan [CVAMP + HDM +PBSCT]). Five single nucleotide polymorphisms were examined based on associations demonstrated in previous individual trials: GSTP1, IL-6, LT-a, TNF-a, and ERCC2 (DNA repair). Patient samples included 412 from E9486, 569 from S9321, and 232 from MRC7 (total=1213). As a combined data set, genotype associations with overall survival did not reach statistical significance, suggesting SNP associations with survival may be therapy specific. One single association that did reach significance in the conglomerate data was TNF-a genotype association with age of disease onset (the high producer variant AA genotype showing a 5 year later age of onset than the AG/GG genotypes, or the overall mean of the trial, age 61 vs 56; p=.03). This is interesting in light of reports suggesting high production of TNF-a is found in myeloma patients and may actually promote myeloma. Yet, high production may also delay disease onset due to its role in other immunologic processes. Additional parameters including genotype associations with bone disease, CR rates, toxicities, and stage are being evaluated and will be presented. Preliminary results show that when SNPs from S9321 were included in a linear regression model, genotype risk groups could be identified; showing 73% correct association with ISS stage 1, and 75% correct association with ISS stage 3. This model, however, was not validated in the E9486 trial, with less than 50% genotype predictive ISS stage association. A number of highly significant ethnic genotype associations were noted, and are presented in a separate abstract. Given the likely complex interactions of multiple genetic variations, collective genotype comparisons may be required from larger pooled data sets BOAC is assembling to increase significant associations.

Abstract Bank On A CureTM is a collaborative project initiated with the International Myeloma Foundation to develop a DNA bank through international cooperation. The Bank provides a resource for the development of cooperative partnerships to assess genetic associations with disease etiology and outcomes. It is well established that genetic variation can have a significant ethnic association, and such variations may account for differences in disease incidence and outcomes. Indeed, African Americans have historically shown a 2.5–3 fold increase population incidence of myeloma compared to the Caucasian population. We have identified a number of potentially biologically relevant genetic variants that show significant differences in ethnic distribution. Using both clinical trials as well as the Coriel [normal] cell line data base, we find in all three cases the same highly significant differences (Fishers Exact Test) between African Americans and Caucasians in functionally relevant genetic polymorphisms, as represented by E9486 univariate analysis of IL-6 (p<.00001), LT-a (p<.0001), ERCC312 (p<.0001), XRCC399 (p=.006), IL-1RA (p<.00001), GST-M (p=.0008), and ERCC751 (p<.02). Subsequently, from trial S9321 we have found differences in a number of clinical outcomes associated with ethnicity. When toxicities between Caucasians and African Americans were examined, similar severe toxicity rates were noted after VAD induction related to neuropathies, nausea, and bone pain; whereas statistically significant differences were noted for infection rates, hyperglycemia, fatigue, dyspnea, and thrombosis (p<.04). During high dose melphalan treatment toxicities were not statistically different. Because significant association between IL-6 genotype and ethnicity was noted, we also examined serum levels of sIL-6R and CRP, available from trial E9486. African Americans patients show significantly lower sIL-6R levels compared to Caucasian patients (mean 149 vs 213 ng/ml; p<.0001). Interestingly, there is a significant association between IL-6 SNP genotype and sIL-6R distribution, with the high IL-6 producer GG genotype (in 90% African Americans) having the lowest sIL-6R compared to the CC or CG genotypes (p=.02). Regression analysis of IL-6 SNP and ethnicity on sIL-6R reveals that ethnicity is significantly associated with sIL-6R distribution after adjustment for IL-6 SNP genotype (p=.001) but not vice versa (p=.91), indicating the association of IL-6 SNP genotype with sIL-6R is due to its association with ethnicity. Together the data demonstrate that while associations may result from co-segregation of ethnic genotypes, they may influence disease risk and outcomes related to important genetic factors distributed unequally in different ethnic populations. Such differences may, for example, alter IL-6 responses. Further, multivariate analysis will be developed. Validation of genotype associations found in different ethnic populations will be an important consideration in evaluating myeloma etiology and outcomes.

17516 Background: The p53 tumor suppressor is commonly inactivated by mutations. Even in tumors without mutations, there are defects in the response to p53 activation. In MM, the prognostic significance of p53 mutation is unknown, while there has been no systematic study of p53 function. We seek to address these issues in this study. Methods: p53 mutation was studied by conformation sensitive gel electrophoresis with primers encompassing exons 1 to 10 followed by sequencing of DNA fragments with altered electrophoretic pattern in newly diagnosed MM patients entered into ECOG E9486 trial where patients were randomized to receive variations of melphalan-based conventional chemotherapy (VBMCP). Fisher’s exact tests were used to compare variables between patients. Kaplan-Meier survival curves were compared using the log-rank test. To investigate p53 function, we analyzed the expression of p53, and 3 of its transcriptional targets, APAF1, p21 and MDM2, in a separate cohort of 15 normal plasma cells (PC), 14 MGUS, 13 smoldering myeloma (SMM) and 101 MM (73 new and 28 relapsed) from the Mayo Clinic who had gene expression profiling performed on the Affymetrix U133A chip (Santa Clara, Ca). Results: Two hundred and sixty-eight patients had enough materials for study and were included in the analysis. The prevalence of p53 mutation was 3% (n = 9). Five of the 9 patients (56%, p = 0.001) with mutations also had p53 deletion (studied by fluorescent in-situ hybridization) resulting in bi-allelic loss of p53. Soft tissue plasmacytomas (37% v 7%, p = 0.018), and among the common translocations, t(4;14) and t(14;16) (50% v 18%) were more common in patients with p53 mutations. Despite similar response to treatment, those with p53 mutation had very short OS (16.7 v 41.4 months, p < 0.001). There was induction of p53 expression in MGUS and SMM with concurrent induction of APAF1, p21 and MDM2 whereas loss of this pattern was frequent in MM (45% in new MM and 60% in relapse MM compared to 15% in MGUS/SMM (p = 0.03)). Conclusions: p53 mutations are relatively rare in newly diagnosed MM patients but portend a short survival. However, functional abnormalities of p53 are prevalent and may be important in progression from MGUS/SMM to MM. [Table: see text]

Abstract We studied the prognostic significance of plasmablastic (PB) multiple myeloma (MM) in Eastern Cooperative Oncology Group Phase III trial E9486. Two reviewers independently reviewed 453 cases. They agreed on 37 PB (8.2%) cases and 416 non-PB cases, achieving an 85% concordance (P < .0001). These PB cases had significantly lower hemoglobin and serum albumin levels, higher calcium and β 2-microglobuin levels, and higher percentage BM plasma cells (PC) by immunofluorescence. They had higher bone marrow PC labeling indices, higher serum soluble interleukin-6 receptor (sIL-6R) levels, and a higher probability of ras mutations. Three treatment regimens were used: vincristine, bis-chloro-ethyl nitrosourea (BCNU) melphalan, cyclophosphamide, and prednisone (VBMCP) alone; VBMCP with added cyclophosphamide (HiCy); or recombinant interferon α 2 (rIFNα2). Although the numbers are low, patients with PB had a significantly lower response rate versus non-PB MM when treated with VBMCP (treated, 47.1% v nontreated, 66.5% [P = .015]). Patients with nonresponding PB had a significantly higher progression rate than non-PB cases (30.6%v 11.8% [P < .0001]), especially with VBMCP alone (35.3% v 15.8% [P = .002]), and with added HiCy (37.5% v 9.8% [P < .0001]), but not with added rIFNα2. Event-free and overall survival of PB MM was shorter (median years, 1.1 v 2.7 and 1.9 v 3.7, respectively [P < .0001 for both]). In multivariate analysis, PB classification was also highly prognostic. There is no survival difference between the patients who were classified as PB by both reviewers versus patients classified as PB by only one reviewer. We conclude that PB MM is a discrete entity associated with more aggressive disease and shortened survival. Tumor cell rasmutations and increased sIL-6R may contribute to a higher proliferation rate and reduced survival. There were significant improvements in response and progression with the addition of HiCy and rIFNα2 to VBMCP, but the numbers were small and improved survival could not be shown.

Recent analyses of circulating blood B cells in myeloma have generated controversy concerning the exact levels of these cells and whether they may represent circulating clonal tumor B cells. Previous reports suggested that CD19+ B cells are markedly increased in myeloma patients and that this population shares clonotypic rearrangements with the malignant plasma cell. We studied the numbers of CD19+ B cells by flow cytometry in previously untreated newly diagnosed myeloma patients in Eastern Cooperative Oncology Group (ECOG) phase III trial E9486. There were 628 patients who were eligible for the clinical protocol E9486, but of these 521 were also entered on the companion laboratory study (E9487) and had CD19 data. In comparison with normal controls, the myeloma patients exhibited a marked heterogeneity in the number of circulating CD19+ B cells as detected by flow cytometry. Approximately 20% of patients had significantly increased levels of circulating CD19+ B cells. However, the total CD19+ blood population from myeloma was not significantly different from the median of age-matched, normal controls. Analysis of CD19+ blood cells in relationship to circulating clonal cells was done in 13 myeloma patients using a clonotypic, quantitative allele-specific oligonucleotide-polymerase chain reaction (PCR) assay. No correlation was found between the numbers of CD19+ B cells (range, 5% to 51%) and PCR estimates of the number of clonal cells in the peripheral blood (range, .009% to 3.6%). Low CD19+ B-cell level (<125 μL) was associated with clinical stage III (P = .033). A significant relationship exists between higher levels (≥125/μL) of CD19 cells and longer overall survival (P < .0001). In addition, high CD19 levels also predicted a clinical response and longer event-free survival. There was a strong inverse association between the level of CD19 values at diagnosis and infections within the first 2 months of diagnosis. Importantly, the number of deaths related to infections was significantly greater in the low versus high CD19 group (P < .0202). Also, CD19 is an independent prognostic factor in addition to plasma cell labeling indices, β2 -microglobulin, hemoglobin, and plasmablastic morphology. Patients with infections were more likely to have low levels of CD19+ cells. In summary, higher CD19+ cell levels are a favorable prognostic sign with no apparent relationship to circulating tumor cells. In addition, this analysis strongly suggests that low peripheral blood levels of CD19+ cells are an adverse prognostic sign in myeloma. The CD19+ cell levels in myeloma patients is an important parameter in the overall assessment of these patients.

We studied the prognostic significance of plasmablastic (PB) multiple myeloma (MM) in Eastern Cooperative Oncology Group Phase III trial E9486. Two reviewers independently reviewed 453 cases. They agreed on 37 PB (8.2%) cases and 416 non-PB cases, achieving an 85% concordance (P < .0001). These PB cases had significantly lower hemoglobin and serum albumin levels, higher calcium and β 2-microglobuin levels, and higher percentage BM plasma cells (PC) by immunofluorescence. They had higher bone marrow PC labeling indices, higher serum soluble interleukin-6 receptor (sIL-6R) levels, and a higher probability of ras mutations. Three treatment regimens were used: vincristine, bis-chloro-ethyl nitrosourea (BCNU) melphalan, cyclophosphamide, and prednisone (VBMCP) alone; VBMCP with added cyclophosphamide (HiCy); or recombinant interferon α 2 (rIFNα2). Although the numbers are low, patients with PB had a significantly lower response rate versus non-PB MM when treated with VBMCP (treated, 47.1% v nontreated, 66.5% [P = .015]). Patients with nonresponding PB had a significantly higher progression rate than non-PB cases (30.6%v 11.8% [P < .0001]), especially with VBMCP alone (35.3% v 15.8% [P = .002]), and with added HiCy (37.5% v 9.8% [P < .0001]), but not with added rIFNα2. Event-free and overall survival of PB MM was shorter (median years, 1.1 v 2.7 and 1.9 v 3.7, respectively [P < .0001 for both]). In multivariate analysis, PB classification was also highly prognostic. There is no survival difference between the patients who were classified as PB by both reviewers versus patients classified as PB by only one reviewer. We conclude that PB MM is a discrete entity associated with more aggressive disease and shortened survival. Tumor cell rasmutations and increased sIL-6R may contribute to a higher proliferation rate and reduced survival. There were significant improvements in response and progression with the addition of HiCy and rIFNα2 to VBMCP, but the numbers were small and improved survival could not be shown.

Abstract Background: Bone disease in myeloma occurs as a result of complex interactions between myeloma cells and the bone marrow microenvironment. To date, no studies have evaluated the potential impact of genetic polymorphisms upon and/or within this microenvironment. Patients and Methods: Peripheral blood DNA from 282 patients enrolled in the Total Therapy 2 (TT2) protocol was studied using the previously reported Affymetrix 3k BOAC custom chip to evaluate relevant genetic polymorphisms. DKK1 gene expression and high risk GEP gene signatures were assessed as previously reported (Blood109:4470–4477 and 109:2276–2284 2007). Patients were classified using both full skeletal x-rays and MRI findings. The lower cut-off used was absence of focal abnormalities on x-ray and/or <7 focal lesions on MRI (see JCO25:1121–1128 2007). Results: The top 200 SNPs were first evaluated based upon univariate P values linked to limited or extensive bone disease. The top 50 SNPs with the smallest P values were then selected (eliminating closely linked genes) for recursive partitioning analysis. The recursive partitioning was conducted both with and without the insertion of known biologically relevant SNPs. The pruned tree developed with recursive partitioning proved to be quite stable and incorporated 4 dominant SNPs: rs 3766934 Epoxide hydrolase (EPHX1); sr3783408 MAP kinase; sr 1062637 RNA helicase DDX18; and sr3181366 TNFSF8-TNF-α. No alternate SNP substituted for EPHX1 in the recursive model. The 4 SNP signature of EPHX1 GG, MAP4K5 AG/AA, DDX18 GG/CC plus TNFSF8 CT/TT was highly correlated with the number of MRI lesions: mean 8.66 lesions versus 3.33 for alternate SNPs (P<.001). In the univariate association of standard prognostic factors, 4 SNP signature and 70 and 17 gene models for high risk, the best correlation with bone disease was with the 4 SNPs (P<.0001) followed by the 17 gene GEP model (P=0.05). Using stepwise multivariate regression analysis the best correlation with bone disease was Log2DKK-1 expression (P=.0001) but closely followed by SNPs EPHX1 (P=.0009); MAP4K5 (P=.0071) plus TNFSF8 and serum LDH (P=.02). The various analyses training (2/3) and validation sets were used with assessment of sensitivity and specificity. The microenvironmental 4 SNP signature combined with GEP DKK1 provided the best prediction of myeloma bone disease and overall outcome. Conclusions: This first assessment of genetic polymorphisms linked to myeloma bone disease in myeloma has led to the identification of polymorphisms with both potential biologic relevance and utility in prognostic models of myeloma bone disease including risk stratification for bisphosphonate use. Validation of these findings may allow a search for potential therapeutic targets.

Abstract Genetic variations in patient populations likely contribute to disease progression and therapeutic outcomes. We have begun a systematic approach to examine the association of genetic variations (ie. functional, single nucleotide polymorhisms, SNPs) involved in myeloma growth promotion, metabolic events, drug responses, and DNA repair on clinical outcome in the intergroup trial S9321. This trial tested a single high dose regimen with autologous stem cell support against a conventional dose regimen, with further randomization of responders to maintenance with interferon or not, in newly diagnosed patients with multiple myeloma. ECOG, CALGB and SWOG enrolled 899 patients with newly diagnosed MM to receive VAD induction x 4 cycles followed by randomization to PBSC-supported high dose therapy (HDT) versus standard dose therapy (SDT) of VBMCP, using CTX 4.5 g/m2 + G-CSF for PBSC mobilization in all patients. Responders to VBMCP or HDT were randomized to IFN or no maintenance. Specimens were distributed through ECOG for biologic correlative studies, including candidate SNP analysis. SNP assays have been developed using the Sequenom Mass-extend platform for functional SNPs in IL-6, IL-1, IL-RA, IL-10, TNF, Lta, TGFb, MDR1, MPO, CYP3A4, GST (M, P, T), ERCC2 and XRCC1, and are being evaluated on 803 DNA samples prepared from patients enrolled in S9321. Preliminary findings (n=135) demonstrate functional genetic variants of IL-10 (position -1082), IL-1 (position +3953), TGFb (postion -509), and TNFa (position -308) are showing trends associated with differences in progression free survival; and variants in IL-6 (position -174) are associated with response. Median survival of the IL-10 variants was 31 months for A/A low producer alleles versus 19 months for the G/G high producer alleles (p=.5). For TNFa, 2 cases with the high producer A/A alleles died within a year, while the median survival for the lower producer G/G alleles was 2 years (p=.04). For patients with the high producer C/C/ allele of IL-1 (n=67), median survival was 2 years, versus 5 patients with the T/T low producer alleles that had a median survival greater than 5 years (preliminary p=.29). While these preliminary results are now only suggestive of trends in genetic polymorphisms associated with clinical outcome, completion of the full SNP panel on the entire sample base should provide a extensive association study, and analysis of potential differences in therapy arms of the trial. The full panel and association studies will be presented.ααββααα