Faculty Bibliography

Purpose: Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means.Experimental Design: To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in in vitro experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in in vivo human myeloma xenograft models.Results: Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation.Conclusions: Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma. Clin Cancer Res; 24(10); 2395-407. ©2018 AACRSee related commentary by Parekh, p. 2237.

Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.

Robust establishment of survival in multiple myeloma (MM) and its relationship to recurrent genetic aberrations is required as outcomes are variable despite apparent similar staging. We assayed copy number alterations (CNA) and translocations in 1036 patients from the NCRI Myeloma XI trial and linked these to overall survival (OS) and progression-free survival. Through a meta-anlysis of these data with data from MRC Myeloma IX trial, totalling 1905 newly diagnosed MM patients (NDMM), we confirm the association of t(4;14), t(14;16), t(14;20), del(17p) and gain(1q21) with poor prognosis with hazard ratios (HRs) for OS of 1.60 (P=4.77 × 10^-7), 1.74 (P=0.0005), 1.90 (P=0.0089), 2.10 (P=8.86 × 10^-14) and 1.68 (P=2.18 × 10^-14), respectively. Patients with 'double-hit' defined by co-occurrence of at least two adverse lesions have an especially poor prognosis with HRs for OS of 2.67 (P=8.13 × 10^-27) for all patients and 3.19 (P=1.23 × 10^-18) for intensively treated patients. Using comprehensive CNA and translocation profiling in Myeloma XI we also demonstrate a strong association between t(4;14) and BIRC2/BIRC3 deletion (P=8.7 × 10^-15), including homozygous deletion. Finally, we define distinct sub-groups of hyperdiploid MM, with either gain(1q21) and CCND2 overexpression (P<0.0001) or gain(11q25) and CCND1 overexpression (P<0.0001). Profiling multiple genetic lesions can identify MM patients likely to relapse early allowing stratification of treatment.

The increased number of effective therapies and the wider use of combinations that give deeper remissions have resulted in a reassessment of the goals of myeloma therapy. With the advent of new therapeutic strategies and diagnostic tools, achievement of minimal residual disease (MRD)-negative status has become increasingly important, with some even considering it as the primary endpoint for therapy. The level of MRD that is aimed for is a continuous, rather than an absolute variable, with studies in both transplant-eligible and -noneligible patients showing that the level of MRD achieved is predictive of progression-free survival and overall survival, with an improvement in survival of approximately 1 year for each log-depletion in MRD level. The most widely used methods to assess MRD status include flow cytometry and clonality detection, using next-generation sequencing technologies with sensitivity limits of 1:10^-3 to 1:10^-6 The timing of when to assess MRD depends on the treatment used, as well as the molecular and cytogenetic subgroup of the myeloma itself. It is also becoming clear that the level of MRD negativity, as well as microenvironmental factors, are important prognostically, including the regeneration of normal plasma cells, and the normalization of the immune repertoire. With advances in antibody-based therapy and immunotherapy, the achievement of stable MRD states is now possible for a significant proportion of patients, and is a prerequisite for myeloma cure.

INTRODUCTION/BACKGROUND:Multiple myeloma is a malignant neoplasm characterized by plasma cell proliferation in blood and bone marrow. Approximately two-thirds of the patients with multiple myeloma are >65 years at the time of diagnosis. Patients in this age group often have co-existing cardiovascular diseases. Areas covered: The presence of multiple myeloma adds to the malady by direct deposition of amyloid protein in the heart or via toxicity of chemotherapeutic agents. Cardiac contractile dysfunction, arrhythmias and thromboembolic disorders are the main issues in the management of elderly patients with multiple myeloma. Expert commentary: Assessment of cardiac risk and functional status requires careful evaluation by history, physical examination and imaging studies such as echocardiography and magnetic resonance imaging. The management of cardiovascular disorders in the presence of multiple myeloma is difficult and poses a challenge for the internist, the oncologist, and the cardiologist alike. This review is an overview of the problem of cardiovascular risk in and management of elderly patients with multiple myeloma.

BACKGROUND:KW-2478 is a novel non-ansamycin Hsp90 inhibitor with modest single-agent activity in relapsed/refractory myeloma but which shows synergistic antimyeloma activity with bortezomib (BTZ) in preclinical studies. This study determined the safety, preliminary clinical activity, and pharmacokinetics of KW-2478, an Hsp90 inhibitor, in combination with BTZ in patients with relapsed/refractory multiple myeloma (MM). METHODS:Phase I dose escalation determined the recommended phase II dose (RP2D) of KW-2478 plus BTZ, which was then used during phase II. RESULTS:on days 1, 4, 8, and 11 every 3 weeks. In the efficacy evaluable phase I/II population treated at the RP2D (n=79), the objective response rate was 39.2% (95% confidence interval: 28.4-50.9%), clinical benefit rate 51.9% (40.4-63.3%), median progression-free survival 6.7 (5.9-not reached (NR)) months, and median duration of response 5.5 (4.9-NR) months. In the phase I/II safety population (n=95), the most frequently observed treatment-related grade 3/4 adverse events were diarrhoea, fatigue, and neutropenia (each in 7.4% of patients), and nausea and thrombocytopenia (each in 5.3%). CONCLUSIONS:KW-2478 plus BTZ was well tolerated with no apparent overlapping toxicity in patients with relapsed/refractory MM. The antimyeloma activity of KW-2478 in combination with BTZ as scheduled in this trial appeared relatively modest; however, the good tolerability of the combination would support further exploration of alternate dosing schedules and combinations.

Recent studies suggest that the evolutionary history of a cancer is important in forecasting clinical outlook. To gain insight into the clonal dynamics of multiple myeloma (MM) and its possible influence on patient outcomes, we analyzed whole exome sequencing tumor data for 333 patients from Myeloma XI, a UK phase 3 trial and 434 patients from the CoMMpass study, all of which had received immunomodulatory drug (IMiD) therapy. By analyzing mutant allele frequency distributions in tumors, we found that 17% to 20% of MM is under neutral evolutionary dynamics. These tumors are associated with poorer patient survival in nonintensively treated patients, which is consistent with the reduced therapeutic efficacy of microenvironment-modulating IMiDs. Our findings provide evidence that knowledge of the evolutionary history of MM has relevance for predicting patient outcomes and personalizing therapy.

JAK proteins have been linked with survival and proliferation of multiple myeloma (MM) cells; therefore, JAK inhibition could be a therapeutic strategy for MM. We evaluated JAK1 and JAK2 expression in MM patients and the effects of JAK/STAT pathway inhibition on apoptosis, cell cycle, gene and protein expression in RPMI-8226 and U266 MM cell lines. 57% of patients presented overexpression of JAK2 and 27%, of JAK1. After treatment with ruxolitinib and bortezomib, RPMI-8226 and U266 presented 50% of cells in late apoptosis, reduction of anti-apoptotic genes expression and higher number of cells in SubG0 phase. Co-culture with stromal cells protected RPMI-8226 cells from apoptosis, which was reversed by lenalidomide addition. Combination of ruxolitinib, bortezomib and lenalidomide induced 72% of cell death, equivalent to bortezomib, lenalidomide and dexamethasone, combination used in clinical practice. Many JAK/STAT pathway genes, after treatment, had their expression reduced, mainly in RPMI-8226, with insignificant changes in U266. In this scenario, JAK/STAT pathway could pose as a new therapeutic target to be exploited, since it is constitutively active and contributes to survival of MM tumor cells.

Monoclonal gammopathy of undetermined significance is a pre-malignant precursor of multiple myeloma with a 1% risk of progression per year. Although targeted analyses have shown the presence of specific genetic abnormalities such as IGH translocations, RB1 deletion, 1q gain, hyperdiploidy or RAS gene mutations, little is known about the molecular mechanism of malignant transformation. We performed whole exome sequencing together with comparative genomic hybridization plus single nucleotide polymorphism array analysis in 33 flow-cytometry-separated abnormal plasma cell samples from patients with monoclonal gammopathy of undetermined significance to describe somatic gene mutations and chromosome changes at the genome-wide level. Non-synonymous mutations and copy-number alterations were present in 97.0% and in 60.6% of cases, respectively. Importantly, the number of somatic mutations was significantly lower in monoclonal gammopathy of undetermined significance than in myeloma (P<10^-4) and we identified six genes that were significantly mutated in myeloma (KRAS, NRAS, DIS3, HIST1H1E, EGR1 and LTB) within the monoclonal gammopathy of undetermined significance dataset. We also found a positive correlation with increasing chromosome changes and somatic gene mutations. IGH translocations, comprising t(4;14), t(11;14), t(14;16) and t(14;20), were present in 27.3% of cases and in a similar frequency to myeloma, consistent with the primary lesion hypothesis. MYC translocations and TP53 deletions or mutations were not detected in samples from patients with monoclonal gammopathy of undetermined significance, indicating that they may be drivers of progression to myeloma. Data from this study show that monoclonal gammopathy of undetermined significance is genetically similar to myeloma, however overall genetic abnormalities are present at significantly lower levels in monoclonal gammopathy of undetermined significant than in myeloma.