Michael Kuehl

MicroRNAs regulate critical genes associated with multiple myeloma pathogenesis

Progress in understanding the biology of multiple myeloma (MM), a plasma cell malignancy, has been slow. The discovery of microRNAs (miRNAs), a class of small noncoding RNAs targeting multiple mRNAs, has revealed a new level of gene expression regulation. To determine whether miRNAs play a role in the malignant transformation of plasma cells (PCs), we have used both miRNA microarrays and quantitative real time PCR to profile miRNA expression in MM-derived cell lines (n = 49) and CD138+ bone marrow PCs from subjects with MM (n = 16), monoclonal gammopathy of undetermined significance (MGUS) (n = 6), and normal donors (n = 6). We identified overexpression of miR-21, miR-106b∼25 cluster, miR-181a and b in MM and MGUS samples with respect to healthy PCs. Selective up-regulation of miR-32 and miR-17∼92 cluster was identified in MM subjects and cell lines but not in MGUS subjects or healthy PCs. Furthermore, two miRNAs, miR-19a and 19b, that are part of the miR-17∼92 cluster, were shown to down regulate expression of SOCS-1, a gene frequently silenced in MM that plays a critical role as inhibitor of IL-6 growth signaling. We also identified p300-CBP-associated factor, a gene involved in p53 regulation, as a bona fide target of the miR106b∼25 cluster, miR-181a and b, and miR-32. Xenograft studies using human MM cell lines treated with miR-19a and b, and miR-181a and b antagonists resulted in significant suppression of tumor growth in nude mice. In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis.

Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development.

In multiple myeloma (MM), an incurable B cell neoplasm, mutation or deletion of p53 is rarely detected at diagnosis. Using small-molecule inhibitors of MDM2, we provide evidence that miR-192, 194, and 215, which are downregulated in a subset of newly diagnosed MMs, can be transcriptionally activated by p53 and then modulate MDM2 expression. Furthermore, ectopic re-expression of these miRNAs in MM cells increases the therapeutic action of MDM2 inhibitors in vitro and in vivo by enhancing their p53-activating effects. In addition, miR-192 and 215 target the IGF pathway, preventing enhanced migration of plasma cells into bone marrow. The results suggest that these miRNAs are positive regulators of p53 and that their downregulation plays a key role in MM development.

Patterns of monoclonal immunoglobulins and serum free light chains are significantly different in black compared to white monoclonal gammopathy of undetermined significance (MGUS) patients

Monoclonal gammopathy of undetermined significance (MGUS), the precursor to multiple myeloma, is more common in blacks than whites. The serum free light chain (sFLC) assay is an important prognostic test in MGUS, but no study has evaluated sFLC levels and ratios in black MGUS patients. One‐hundred and twenty‐five black MGUS patients at two urban centers were compared to the white population of the Mayo Clinic. The median age for blacks was 73 years [41–94] and 75% were male. The M‐protein isotype in blacks was 81% IgG, 13% IgA, 2% IgM, and 4% biclonal compared to 70%, 12%, 16%, and 2%, respectively, in whites, (P < 0.0005). The median M‐protein concentration for blacks was 0.44 gm/dL (trace‐2.33) compared to 1.2 gm/dl in whites. An abnormal sFLC ratio was present in 45% of black compared to 33% of white (P = 0.01) patients. Using the Mayo Clinic risk model, black patients had a significantly lower proportion of higher risk MGUS compared to whites: low 43%, low‐intermediate 45%, high‐intermediate 10%, and high 2% (P = 0.014). Black patients with MGUS have significantly different laboratory findings compared to whites. The biologic basis for these disparities and their effect on prognostic assessment is unknown. Prognostic models based on these biomarkers should be used cautiously in nonwhite populations. Am. J. Hematol. 2011.

Smoldering (asymptomatic) multiple myeloma: revisiting the clinical dilemma and looking into the future.

Recent studies show that multiple myeloma (MM) is consistently preceded by an asymptomatic precursor state. Smoldering MM (SMM) is a MM precursor defined by an M-protein concentration >or= 3 g/dL and/or >or= 10% bone marrow plasma cells, in the absence of end-organ damage. Compared with individuals diagnosed with monoclonal gammopathy of undetermined significance (MGUS), patients with SMM have a much higher annual risk of developing MM. However, based on clinical observations, the natural history of SMM varies greatly, from stable MGUS-like disease to highly progressive disease. Using conventional clinical markers, SMM patients can be stratified into 3 risk groups. Importantly, because of considerable molecular heterogeneity, we currently lack reliable markers to predict prognosis for individual SMM patients. Furthermore, until recently, potent drugs with reasonable toxicity profiles have not been available for the development of early MM treatment strategies. Consequently, current clinical guidelines emphasize the application of close clinical monitoring followed by treatment when the patient develops symptomatic MM. This review focuses on novel biomarkers, molecular profiles, and microenvironmental interactions of interest in myelomagenesis. We also discuss how the integration of novel biologic markers and clinical monitoring of SMM could facilitate the development of early treatment strategies for high-risk SMM patients in the future.

Erratum: Downregulation of p53-inducible microRNAs 192, 194, and 215 Impairs the p53/MDM2 Autoregulatory Loop in Multiple Myeloma Development (Cancer Cell (2010) 18(4) (367–381) (S1535610810003429) (10.1016/j.ccr.2010.09.005))

Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development. / Pichiorri F; Suh SS; Rocci A; De Luca L; Taccioli C; Santhanam R; Zhou W; Benson DM Jr; Hofmainster C; Alder H; Garofalo M; Di Leva G; Volinia S; Lin HJ; Perrotti D; Kuehl M; Aqeilan RI; Palumbo A; Croce CM.. In: CANCER CELL. ISSN 1535-6108. 18(2010), pp. 367-381. Original Citation: Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development.

Abstract 5472: Systems pharmacogenomics approach identifies synergistic molecular action of combined MTOR/HDAC inhibition on MYC

Multiple myeloma (MM) and murine plasmacytoma (PCT) are rare mature B-lymphoid malignancies. Allelic variants of Mtor and Cdkn2a affect susceptibility to PCT, and functional alterations in the PI3K/MTOR and CYCLIN/CDK/CDKI/RB (RB) pathways are common to both malignancies. We found that combining sirolimus (rapamycin), an inhibitor of mechanistic target of rapamycin (MTOR), with entinostat (MS-275), a selective class I histone deacytlase (HDAC) inhibitor, was synergistic in controlling 90% of tested cell lines derived from B cell malignancies in vitro, effective in limiting xenograft growth in vivo, and diminished cellular viability in ex vivo patient samples. Similarly, the combination reduced tumor burden and volume and increased survival in a long-term, in-vivo study in C.B6-Bcl2l1 mice. To examine the core synergistic consequence of combining entinostat with sirolimus, an integrated, systems-level approach was used. Weighted gene co-expression analysis (WGCNA) of GEP data from MM cells treated individually and in combination was used to identify a distinct module of 126 genes cooperatively affected by both drugs. Of the cooperatively affected genes, 37 were found to be differentially expressed in MM and predictive of survival (p Citation Format: Benjamin J. Gamache, John K. Simmons, Aleksandra Michalowski, Jyoti Patel, Ke Zhang, Shuling Zhang, Wendy DuBois, Adriana Zingone, Michael Kuehl, Jing Huang, Ola Landgren, Beverly Mock. Systems pharmacogenomics approach identifies synergistic molecular action of combined MTOR/HDAC inhibition on MYC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5472. doi:10.1158/1538-7445.AM2014-5472

Abstract 2217: A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The necessity of combining targeted therapeutics to achieve optimal, lasting clinical benefit is clear, but standardized approaches for identifying the interactive effects of these combinations are not yet established. Discerning drug synergy at the molecular level has proven particularly challenging, yet identification of cooperatively responding, biologically-relevant targets could be useful for defining patient subsets for which the combination would be active. Here we used a transcriptional co-expression systems-level analysis to define the cooperative molecular response to the synergistic combination of mTOR/HDAC inhibitors in multiple myeloma (MM), and in other tumor types including triple negative breast cancer. Co-expression analysis of cells treated individually and in combination defined the contribution of each drug to the combination, and identified a distinct network of 126 genes cooperatively targeted by both drugs. We interrogated the cooperative network genes for differential expression between normal and malignant cells, as well as for correlation with survival in a large patient dataset. 37 of the cooperatively affected genes were both differentially expressed in MM and predictive of survival (p<0.01). Analysis of additional tumor types showed similar results. The pharmacodynamic response of the survival-linked signature to the drug combination was evaluated using the NanoString gene expression platform in a large number of cell lines from multiple tumor types and in ex vivo-treated primary patient samples before and after treatment. We found the expression change of signature genes to be highly specific for biological response to the drug combination across tumor types. Additionally, to link the response signature to a central molecular effect of combination treatment, Ingenuity transcription factor enrichment testing was performed. Based on these predictions, subsequent analysis of CHIP-Seq datasets was performed, and two oncogenic transcription factors (TFs) were found to bind nearly all genes of this signature. We then experimentally linked drug combination response to diminished expression of these TFs at the protein level ahead of cell cycle and apoptotic changes. Further experiments have been performed to establish a direct link between these TFs, our gene signature, and drug response. Thus, a systems-level genomic approach has identified a gene signature indicative of drug combination activity, mechanism, disease specificity, and clinical potential. Citation Format: John K. Simmons, Aleksandra M. Michalowski, Ben Gamache, Jyoti Patel, Adriana Zingone, Ke Zhang, Michael Kuehl, Jing Huang, Ola Landgren, Beverly A. Mock. A systems pharmacogenomic approach to identify synergistic molecular mechanisms of combined mTOR/HDAC inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2217. doi:10.1158/1538-7445.AM2013-2217

Abstract 219: TP53 loss: An overriding marker of disease progression in Multiple Myeloma

Multiple Myeloma (MM) is an incurable plasma-cell disorder progressing from indolent monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) to relapse MM and finally plasma cell leukemia (PCL). Alterations affecting TP53 are among the principal genetic progression events associated with disease progression. We analyzed results from gene expression profiling (GEP), array-based comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH) and mutational data on the largest cohort published to date of 806 patients from all MM stages and 48 human myeloma cell lines (HMCLs). The prevalence of monoallelic TP53 deletions by aCGH is 1.4%, 3.7%, 8.9% and 25%, in MGUS, SMM, newly diagnosed and relapsed MM respectively. It reaches 53% in HMCLs and 75% in secondary PCL. No biallelic TP53 deletion were observed in patients and only 8% in HMCLs. In relapsed MM, the prevalence of TP53 deletions increases from 12/75 patients (16%) at first, to 9/31 patients (29%) in second and 5/7 patients (71%) in third relapse or more. We studied by FISH the status of TP53 in 113 patients from early stages progressing to relapse. Overall 15/113 patients had TP53 deletion, we confirmed that 10/15 patients acquired TP53 deletion at late stages. Next, we investigated the mutational status of TP53 in 59 relapsed MM patients, 5/59 samples (8.5%) had TP53 mutations, 4/5 mutations were located in the DNA binding domain. Presence of TP53 deletion conferred poor overall survival in relapsed MM patients (4.2 vs. 37.8 months, p = 0.015). A cohort of 239 patients had GEP-aCGH paired analysis we used a validated 70-gene model to stratify patients based on calculations obtained by GEP. The cohort was divided into quartiles and we observed the prevalence of TP53 in the low and high-risk groups. TP53 deletion was observed in 33/239 patients (14%) by aCGH, 2/33 (6%) had GEP values in the low-risk and 13/33 (39%) in the high-risk group. Next, we used a previously proposed cut-off value predicting TP53 deletion when GEP values are less than 733. We tried to validate this value as a surrogate for direct detection of TP53 deletion, and used the positive predictive value (PPV) of such level as a potential clinically useful marker. The fact that aCGH is the gold standard approach to define copy-number alterations, the proposed cut-off value failed to predict TP53 deletion as the PPV was low at 18% and the sensitivity of 69%. Finally, we studied by aCGH the status of MDM2 and CDKN2A, two key regulators of TP53. MDM2 gain and CDKN2A loss were infrequent events in patients even in advanced disease. In 48 HMCLs, only one had MDM2 copy gain (2%) while 14 cell lines had CDKN2A loss (29%). In MM, emergence of TP53 deletion/mutations in MM and its increase in aggressive stages of disease remain the overriding genetic factor for poor prognosis determination and a marker of progressive genetic events. Based on GEP, the 733 cut-off value did not accurately predict TP53 deletion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 219. doi:10.1158/1538-7445.AM2011-219