scholarly journals Delineating CDK9 Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1598-1598
Author(s):  
Osman Aksoy ◽  
Judith Lind ◽  
Vincent Sunder-Plassmann ◽  
Martin Percherstorfer ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Microenvironment ◽  
Tumor Cell ◽  
Treatment Strategies ◽  
Loss Of Function ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Protein Levels ◽  
Molecular Events

Abstract Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, but not other genes (e.g. Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 induced a reduction of mRNA/ protein levels of Mcl-1, cMyc, RNA Pol II, but not of other potential targets (e.g. Bcl-2) in a dose- and time-dependent manner. Moreover, Thal-sns-032 reduced Mdm2 and thereby increased p53 protein levels. Consequently, Thal-sns-032 inhibited tumor cell proliferation and survival both in tumor cell- and tumor cell:BMSC co- cultures. Rationally, derived combination strategies of Thal-sns-032 for example with venetoclax, but also other investigational and established MM therapies induced synergistic anti-MM effects within the tumor microenvironment. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derive MM combination treatment strategies. Disclosures Vallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

Mir-29a Displays in Vitro and in Vivo Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2090-2090
Author(s):  
Manujendra N Saha ◽  
Yan Chen ◽  
Jahangir Abdi ◽  
Hong Chang
Keyword(s):  
Multiple Myeloma ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Treatment Strategies ◽  
Loss Of Function

Abstract Despite advances in recent therapeutic approaches including targeted therapies, multiple myeloma (MM) remains still incurable necessitating the development of novel treatment strategies. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. In this study, we sought to characterize the role of miR-29a as a tumor suppressor as well as evaluated its therapeutic potential in MM. miR-29a expression levels were found down-regulated in a panel of 5 MM cell lines, 6 newly diagnosed MM patient samples compared to its expression in normal hematopoietic cells collected from 10 normal healthy individuals suggesting that high expression of miR-29a might be involved in MM pathogenesis. We further assessed the functional significance of miR-29a by both gain- and loss-of-function studies. A significant decrease in cell viability (22-32%, p<0.05), along with induction of apoptosis (30-35%, p<0.05) was observed at 48 hrs in MM cell lines, MM.1S and 8226 transfected with miR-29a compared to cells transfected with scrambled miRNA. In contrast, cell lines transfected with miR-29a antagonist prevented the loss of viability in such cells indicating the specificity of miR-29a. At the molecular level, we have identified c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, as a target of miR-29a. Binding site of miR-29a was first identified by computer algorithm and further confirmed by the use of a 3’UTR of c-Myc reporter (luciferase renilla/firefly) constructs containing, miR-29a target site. Moreover, treatment with PRIMA-Met, a small molecule anti-tumor agent in phase I/II clinical trials, significantly increased the expression of miR-29a (2 to 6-fold) and decreased expression of c-Myc in MM cell lines and primay MM patient samples suggesting an important role of miR-29a in inhibiting proliferation of MM cells. On the other hand, overexpression of c-Myc in 8226 and MM.1S cells at least partially reverted the functional effect of miR-29a or PRIMA-1Metsuggesting a specific role of c-Myc in mediating its anti-proliferative activity. To examine therapeutic potential of our studies, we took advantage of novel lipid based delivery method of miRNA. Intratumor delivery of the miR-29a by intraperitoneal injection route against MM xenografts in SCID mice resulted in a significant inhibition of tumor growth (~60%) at 12 days of treatment and prolongation of survival (median survival increased from 22 days to 35 days, p<0.038) compared to the mice receiving scrambled miRNA. Retrieved tumors from treated mice showed efficient increase in miR-29a (5.5-fold, p=0.025), and decrease in c-Myc protein as well as reduced expression of Ki67 and increase of Tunel expression. Similar phenomenon was observed by systematic delivery of miR-29a (by intraveneous injection) in mice with no significant side effects or toxicity in mice. Our study reveals an important role of miR-29a as a tumor suppressor in mediating anti-tumor activities in MM cells by targeting c-Myc. Our findings provide a proof-of-principle that formulated synthetic miR-29a exerts therapeutic activity in preclinical models, and support a framework for development of miR-29a based treatment strategies in MM patients. Disclosures No relevant conflicts of interest to declare.

Overcoming p53 Loss with a Multi-Targeted Small Molecule Inhibitor in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4915-4915
Author(s):  
Diana Cirstea ◽  
Teru Hideshima ◽  
Loredana Santo ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Atp Depletion ◽  
Advisory Committees ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Molecule Inhibitor ◽  
Rpmi 8226

Abstract Abstract 4915 Multiple studies have highlighted the critical role of mutation and loss of p53 function in multiple myeloma (MM) when acquiring a more aggressive phenotype and refractoriness to treatment. Therefore, agents capable of overcoming p53 mutational status are important in the context of MM therapeutics. We have previously reported the in vitro and in vivo anti-MM activity of the multi-targeted small molecule inhibitor RGB-286638. Using a human MM cell xenograft model in SCID mice we demonstrated that RGB-286638 inhibited tumor growth and prolonged survival. Our data confirmed suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes in MM.1S MM cells containing wt-p53, which was correlated with rapid downregulation of Rb phosphorylation, resulting in effective G2/M cell cycle blockage and increased sub-G1phase. RGB-286638 induced dose and time-dependent inhibition of RNA pol II phosphorylation as an early event promptly followed by p53 induction. Moreover, RGB-286638 treatment was associated with p53 phosphorylation at ser 15, indicative of DNA damage followed by apoptosis, evidenced by caspases 8, 9 and 3 cleavage and confirmed by Annexin V/PI staining. All together these data suggested that RGB-286638-induced RNA pol II inhibition triggers cytotoxicity in MM cells via p53-dependent apoptosis. Interestingly, RGB-286638 demonstrated cytotoxic activity even in p53-deficient conventional drug-resistant RPMI 8226/Dox 40 MM cells. RGB-286638 treatment of RPMI 8226/Dox40 MM cells showed increased PARP response associated with enhanced NAD depletion followed by increased ATP consumption. Furthermore, concomitant assessment of RGB-286638-induced ATP depletion versus cytotoxicity demonstrated more than 60% ATP loss preceded cell death in RPMI 8226/Dox40 but not in MM.1S. This data suggests the role of either p53-mediated apoptosis (when active) or PARP-induced NAD/ATP depletion and bioenergetic crisis (when absent). Interestingly, the knockdown of p53 did not rescue MM.1S cells from RGB 286638-induced death, suggesting the existence of alternative p53-independent pathways through which RGB-286638 exerts its cytotoxic activity. Ongoing studies are addressing the molecular effects of p53 silencing in MM cells. In addition, dissecting the mechanism of RGB-286638 p53-independent cytotoxicity in MM cells will provide insights for future therapeutic strategies in patients with aggressive MM and associated mutated/deleted-p53. Disclosures Loferer: GPC Biotech AG: Employment. Munshi:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Raje:Celgene: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding.

scholarly journals Transient Receptor Potential C 1/4/5 Is a Determinant of MTI-101 Induced Calcium Influx and Cell Death in Multiple Myeloma

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1490
Author(s):  
Osama M. Elzamzamy ◽  
Brandon E. Johnson ◽  
Wei-Chih Chen ◽  
Gangqing Hu ◽  
Reinhold Penner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Transient Receptor Potential ◽  
Cyclic Peptide ◽  
Calcium Influx ◽  
Acquired Resistance ◽  
Treatment Strategies ◽  
Prognostic Indicators ◽  
Causative Role

Multiple myeloma (MM) is a currently incurable hematologic cancer. Patients that initially respond to therapeutic intervention eventually relapse with drug resistant disease. Thus, novel treatment strategies are critically needed to improve patient outcomes. Our group has developed a novel cyclic peptide referred to as MTI-101 for the treatment of MM. We previously reported that acquired resistance to HYD-1, the linear form of MTI-101, correlated with the repression of genes involved in store operated Ca2+ entry (SOCE): PLCβ, SERCA, ITPR3, and TRPC1 expression. In this study, we sought to determine the role of TRPC1 heteromers in mediating MTI-101 induced cationic flux. Our data indicate that, consistent with the activation of TRPC heteromers, MTI-101 treatment induced Ca2+ and Na+ influx. However, replacing extracellular Na+ with NMDG did not reduce MTI-101-induced cell death. In contrast, decreasing extracellular Ca2+ reduced both MTI-101-induced Ca2+ influx as well as cell death. The causative role of TRPC heteromers was established by suppressing STIM1, TRPC1, TRPC4, or TRPC5 function both pharmacologically and by siRNA, resulting in a reduction in MTI-101-induced Ca2+ influx. Mechanistically, MTI-101 treatment induces trafficking of TRPC1 to the membrane and co-immunoprecipitation studies indicate that MTI-101 treatment induces a TRPC1-STIM1 complex. Moreover, treatment with calpeptin inhibited MTI-101-induced Ca2+ influx and cell death, indicating a role of calpain in the mechanism of MTI-101-induced cytotoxicity. Finally, components of the SOCE pathway were found to be poor prognostic indicators among MM patients, suggesting that this pathway is attractive for the treatment of MM.

scholarly journals DNA Damage Response in Multiple Myeloma: The Role of the Tumor Microenvironment

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 504
Author(s):  
Takayuki Saitoh ◽  
Tsukasa Oda
Keyword(s):  
Multiple Myeloma ◽  
Dna Damage ◽  
Dna Repair ◽  
Tumor Microenvironment ◽  
Dna Damage Response ◽  
Genetic Instability ◽  
Dna Repair Mechanisms ◽  
Damage Response ◽  
Repair Mechanisms

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM cells present various forms of genetic instability, including chromosomal instability, microsatellite instability, and base-pair alterations, as well as changes in chromosome number. The tumor microenvironment and an abnormal DNA repair function affect genetic instability in this disease. In addition, states of the tumor microenvironment itself, such as inflammation and hypoxia, influence the DNA damage response, which includes DNA repair mechanisms, cell cycle checkpoints, and apoptotic pathways. Unrepaired DNA damage in tumor cells has been shown to exacerbate genomic instability and aberrant features that enable MM progression and drug resistance. This review provides an overview of the DNA repair pathways, with a special focus on their function in MM, and discusses the role of the tumor microenvironment in governing DNA repair mechanisms.

scholarly journals Pan-cancer analysis reveals tumor-associated macrophage communication in the tumor microenvironment

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Linbang Wang ◽  
Tao He ◽  
Jingkun Liu ◽  
Jiaojiao Tai ◽  
Bing Wang ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cell ◽  
Hub Genes ◽  
Tumor Associated Macrophage ◽  
Molecular Features ◽  
Receptor Interactions ◽  
Tumor Types ◽  
Pan Cancer

Abstract Background Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment (TME). However, their contribution to the immunosuppressive status of the TME remains unclear. Methods We integrated single-cell sequencing and transcriptome data from different tumor types to uncover the molecular features of TAMs. In vitro experiments and prospective clinical tests confirmed the results of these analysis. Results We first detected intra- and inter-tumoral heterogeneities between TAM subpopulations and their functions, with CD86+ TAMs playing a crucial role in tumor progression. Next, we focused on the ligand-receptor interactions between TAMs and tumor cells in different TME phenotypes and discovered that aberrant expressions of six hub genes, including FLI1, are involved in this process. A TAM-tumor cell co-culture experiment proved that FLI1 was involved in tumor cell invasion, and FLI1 also showed a unique pattern in patients. Finally, TAMs were discovered to communicate with immune and stromal cells. Conclusion We determined the role of TAMs in the TME by focusing on their communication pattern with other TME components. Additionally, the screening of hub genes revealed potential therapeutic targets.

scholarly journals Pursuing a Curative Approach in Multiple Myeloma: A Review of New Therapeutic Strategies

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2015 ◽  
Author(s):  
Mattia D'Agostino ◽  
Luca Bertamini ◽  
Stefania Oliva ◽  
Mario Boccadoro ◽  
Francesca Gay
Keyword(s):  
Multiple Myeloma ◽  
Residual Disease ◽  
Treatment Strategies ◽  
Progression Free Survival ◽  
Hematologic Cancer ◽  
Focus On Results ◽  
Individualize Treatment ◽  
New Generation ◽  
Effective Drugs

Multiple myeloma (MM) is still considered an incurable hematologic cancer and, in the last decades, the treatment goal has been to obtain a long-lasting disease control. However, the recent availability of new effective drugs has led to unprecedented high-quality responses and prolonged progression-free survival and overall survival. The improvement of response rates has prompted the development of new, very sensitive methods to measure residual disease, even when monoclonal components become undetectable in patients’ serum and urine. Several scientific efforts have been made to develop reliable and validated techniques to measure minimal residual disease (MRD), both within and outside the bone marrow. With the newest multidrug combinations, a good proportion of MM patients can achieve MRD negativity. Long-lasting MRD negativity may prove to be a marker of “operational cure”, although the follow-up of the currently ongoing studies is still too short to draw conclusions. In this article, we focus on results obtained with new-generation multidrug combinations in the treatment of high-risk smoldering MM and newly diagnosed MM, including the potential role of MRD and MRD-driven treatment strategies in clinical trials, in order to optimize and individualize treatment.

scholarly journals A Review of Key Biological and Molecular Events Underpinning Transformation of Melanocytes to Primary and Metastatic Melanoma

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 2041 ◽  
Author(s):  
Louise A. Jackett ◽  
Richard A. Scolyer
Keyword(s):  
Treatment Strategies ◽  
Genomic Analysis ◽  
Molecular Therapy ◽  
Health Concern ◽  
Targeted Molecular Therapy ◽  
Public Health Concern ◽  
Molecular Events ◽  
Key Events ◽  
Potential Tool

Melanoma is a major public health concern that is responsible for significant morbidity and mortality, particularly in countries such as New Zealand and Australia where it is the commonest cause of cancer death in young adults. Until recently, there were no effective drug therapies for patients with advanced melanoma however significant advances in our understanding of the biological and molecular basis of melanoma in recent decades have led to the development of revolutionary treatments, including targeted molecular therapy and immunotherapy. This review summarizes our current understanding of the key events in the pathway of melanomagenesis and discusses the role of genomic analysis as a potential tool for improved diagnostic evaluation, prognostication and treatment strategies. Ultimately, it is hoped that a continued deeper understanding of the mechanisms of melanomagenesis will lead to the development of even more effective treatments that continue to provide better outcomes for patients with melanoma.

A Phase 1 Trial of SNS-032, a Potent and Specific CDK 2, 7 and 9 Inhibitor, in Chronic Lymphocytic Leukemia and Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3178-3178 ◽  
Author(s):  
William G. Wierda ◽  
R. Chen ◽  
William Plunkett ◽  
Steven Coutre ◽  
Ashraf Z. Badros ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Chronic Lymphocytic Leukemia ◽  
Dose Escalation ◽  
Phase 1 ◽  
Preliminary Evidence ◽  
Lymphocytic Leukemia ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Survival Factors

Abstract SNS-032, formerly BMS-387032, is a highly selective and potent inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9. CDK2 and CDK7 are involved in cell cycle regulation. CDK7, along with CDK9, regulate RNA polymerase (Pol) II-dependent transcription. Temporary inhibition of RNA Pol II-dependent transcription by SNS-032 has significant effects on short half-life transcripts and proteins, particularly survival factors, cell cycle regulatory proteins, and cytokines that are critical for the survival of malignant B-cells in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). A phase 1 dose-escalation study in patients with MM and CLL is ongoing with separate dose escalations for each indication. The study is designed to evaluate safety, pharmacokinetics (PK) and preliminary evidence of activity of a loading dose (LD) followed by a 6 hour infusion of SNS-032 given weekly for 3 consecutive weeks of each 28-day cycle. Dose and schedule aim to maintain for 6 hours threshold plasma concentrations of 115 ng/mL (in vitro IC90) and higher. The study incorporates an exploratory analysis of potential pharmacodynamic (PD) biomarkers such as decreased phosphorylation of RNA Pol II C-terminal domain to demonstrate inhibition of CDK7 and CDK9, and decreased expression of survival factors to indicate transcriptional inhibition. Methods: Previously treated patients with advanced CLL or MM, measurable disease, and ECOG status 0–1 were eligible. Increasing doses of SNS-032 given as an LD followed by a 6 hr infusion were evaluated. The total starting dose was 15 mg/m2 comprised of a LD of 5 mg/m2 followed by 10 mg/m2 over 6 hr with dose escalation by modified Fibonacci. PD studies of target modulation were performed on peripheral blood mononuclear cells (PBMC) obtained pre- and post-dose. Direct target modulation or downstream effects of target inhibition were evaluated. Results: 35 patients have been treated to date, 18 MM patients and 17 CLL patients. Median age was 61 (range 45–82), with 13 females and 24 males. Median number of prior therapies was 5 (range: 1–11). MM patients have received total doses of 15 – 75 mg/m2. No drug-related dose limiting toxicities (DLTs) or objective responses have been reported thus far in MM. CLL patients have received total doses of 15 –100 mg/m2. No drug-related DLTs were observed through the 50 mg/m2 dose cohort. At 75 mg/m2, concentrations of SNS- 032 exceeded IC90 (mean maximum concentration during the 6 hr infusion was 261 ± 45 ng/mL). Evidence of biochemical tumor lysis syndrome (TLS) was observed in all CLL patients treated at this dose level. One patient experienced a DLT of vascular leak syndrome and failure to receive all 3 cycle 1 doses. One CLL patient has been treated thus far at 100 mg/m2. This patient experienced TLS with a DLT of elevated liver function enzymes for &gt;48 hr and received only 2 of 3 doses in cycle 1. No objective responses have been observed. PD analyses showed evidence of decreased Mcl-1 or XIAP in several patients. Conclusions: The mechanism of action of SNS-032 supports testing this agent in B-cell malignancies such as MM and CLL. A pharmacologically-derived dose regimen that sustains IC90 SNS-032 concentrations or higher for 6 hr is being studied; target levels were achieved and exceeded in cohort 5 (75 mg/m2) for both MM and CLL. No DLTs or objective responses have been observed thus far in MM. AEs and DLTs related to mild to moderate TLS were observed in CLL patients at 75 mg/m2 and higher. No objective responses have been observed. Preliminary evidence of target-specific PD modulation has been demonstrated. Enrollment in this trial is continuing.

Identification of Rab5 as a Gene Involved in Chronic Myeloid Leukemia (CML) Progression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3470-3470
Author(s):  
Daniela Cilloni ◽  
Monica Pradotto ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Enrico Bracco ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Small Gtpases ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Protein Levels

Abstract Abstract 3470 Poster Board III-358 The role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, however, the mechanisms leading to CML progression remain poorly understood. By using our model of transgenic Drosophila Melanogaster (Dm) for human Bcr-Abl driven CML we have identified Rab5 as a gene involved in the regulation of CML progression. The Rab5 is a member of gene family small GTPases which are involved in the regulation of vesicular transport. Lately several important reports have linked some members of the Rab family to invesivness and migration of cancer cells. Rab5 is associate with alpha-integrin subunits and modulates their endosomal traffic and subcellular localization. We have observed that a loss of function of Rab5 gene have induced a worsening of the CML phenotype generated by hBcr-Abl expression. In contrast, Rab gain of function rescued Bcr-Abl phenotype. The aim of the study was to evaluate the expression of Rab5 in CML cells to better understand if a potential correlation with progression, which has been observed in the model, could be confirmed in patients. Methods Rab5 gene expression was measured by Real Time PCR in 90 samples from 80 CML patients (32 PB and 58 BM). Among those, 53 are collected at diagnosis (19 of 53 patients have been enrolled in TOPS study). In addition, 9 samples from in CP patients have been collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC. In 14 patients, genes expression was analyzed during remission as, well. In parallel, 21 healthy donors (10 PB and 11 BM) have been evaluated. Rab5 protein expression was investigated by Western Blot and Immunofluorescence. We have also utilized K562 transfected with Rab5 plasmid, which we have generated to gain insight about the effects of Rab5 on cell proliferation and apoptosis. Results Rab5 transfection and overexpression in K562 significantly reduced proliferation and affected apoptosis. We found that in CML patients Rab5 expression levels were significantly decreased in either BM or PB (p<0.001 and p<0.0001) as compared to healthy subjects. Furthermore, in blast crisis samples we have found Rab5 transcripts levels to be further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Our preliminary analysis of samples from TOPS trial have shown a trend that Rab5 levels are lower among those patients achieving MMR by 12 months, when compared to the group of patients non achieving MMR on 400 mg, but that difference was not statistically significant (p=0.2). Among those randomized to receive imatinib 800 mg the difference was statistically significant with a median value among those achieving MMR of 1.27 vs 2.14 in the group without MMR (p=0.04). The protein levels have been analyzed by Western Blot and immunofluorescence and allow us to show detectable levels of Rab5 in samples collected at remission, but undetectable levels in course of active CML disease. Although preliminary, our results show a significant decrease of Rab5 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Rab5 in slowing down or suppressing a progression. Surprisingly, among CP CML patients the responders to TKI therapy have been detected to express a lower level of Rab5 than non responders. We are conducting further studies to better explain these data, which we find intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network Disclosures No relevant conflicts of interest to declare.

Disruption Of Super Enhancer-Driven Cancer Dependencies In Diffuse Large B-Cell Lymphoma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3021-3021 ◽  
Author(s):  
Bjoern Chapuy ◽  
McKeown Michael ◽  
Charles Y. Lin ◽  
Stefano Monti ◽  
Margaretha GM Roemer ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Dana Farber Cancer Institute ◽  
Super Enhancer ◽  
Equity Ownership

Abstract Diffuse large B-cell lymphoma (DLBCL) exhibits significant biological and transcriptional heterogeneity which is conferred, in part, by pathologic modulation of lineage-specific and growth-associated master regulatory transcription factors (TF). Chromatin associated with TF binding sites is markedly enriched in histone proteins that are post-translationally modified by lysine side-chain acetylation. This mark facilitates the opening of chromatin and recruits a class of co-activators which recognize ε-acetyl lysine through a bromodomain. The sub-family of bromodomain and extra-terminal domain (BET) co-activators (BRD2, BRD3 and BRD4) are appealing, in part, because transgenic expression of BRD2 caused a DLBCL-like neoplasm in mice. We recently developed the first BET inhibitor, JQ1, and now explore the role of BET bromodomains in oncogenic transcription and assess BET family members as therapeutic targets in DLBCL. Nanomolar doses of JQ1 and 3 structurally dissimilar BET bromodomain inhibitors decreased the cellular proliferation of a broad panel of DLBCL cell lines of all transcriptionally defined types whereas the inactive enantiomer, JQ1R, had no effect. BRD2 and BRD4 depletion similarly decreased the proliferation of multiple DLBCL cell lines. We next explored the therapeutic potential of BET inhibition in two independent DLBCL xenotransplantation models, Ly1 and Toledo. In the first xenograft model, JQ1-treated mice had a prolongation of overall survival (p = 0.003). In the second model, JQ1-treated animals had significantly delayed tumor progression and decreased lymphomatous infiltration of spleen and bone marrow. To define the transcriptional pathways regulated by BET bromodomain proteins, we performed transcriptional profiling of multiple vehicle and JQ1-treated DLBCL cell lines. Following JQ1 treatment, we observed downregulation of multiple MYD88/TLR and BCR signaling pathway components and functionally validated MYC and E2F target gene sets. BET inhibition decreased MYC transcripts and protein in the DLBCL cell line panel suggesting that BET bromodomains directly modulate MYC transcription. In contrast, JQ1 treatment did not measurably alter E2F1 transcript or protein abundance suggesting a co-activator role of the BET bromodomains for E2F1. To explore the role of BET bromodomains in oncogenic E2F1 transcriptional signaling, we performed ChIPSeq experiments in Ly1 cells, using a chemical genetic approach. Rank-ordering of all transcriptionally active promoters based on H3K4me3 enrichment and RNA Pol II occupancy identifies pervasive binding and spatial colocalization of BRD4 and E2F1 to active promoter elements. We identified a JQ1-mediated transcriptional elongation defect across E2F1-bound promoters, responsible for the downregulation of E2F1 targets. As oncogenic TFs may signal to RNA Pol II through distal enhancer elements, we also characterized the genome-wide localization of BRD4 to enhancers in the Ly1 DLBCL cell line. Rank-ordering of enhancer regions by H3K27ac enrichment reveals that BRD4 binds to the vast majority of active enhancers in the Ly1 genome. Strikingly, the BRD4 load is asymmetrically distributed throughout the genome at enhancer sites with only a small subset of BRD-loaded “super enhancers (SE)”, 285/18330 (1.6%), accounting for 32% of all BRD4 enhancer binding in the cell. The POU2AF1 locus emerged as the most BRD4-overloaded enhancer in Ly1. BET inhibition reduced RNA Pol II elongation of POU2AF1, with a concomitant increase in promoter-paused RNA Pol II near the transcriptional start site. Accordingly, JQ1 treatment decreased POU2AF1 transcript abundance and protein expression and reduced the expression of a POU2AF1 target gene set. POU2AF1 depletion with independent shRNAs significantly decreased the proliferation of Ly1 and enforced POU2AF1 expression decreased the sensitivity of Ly1 cells to JQ1 treatment. Additional super enhancer-driven genes that were sensitive to JQ1 treatment include ones which promote and maintain the B-cell gene expression program and limit plasma cell differentiation. Our data suggest that BET inhibition limits the growth of DLBCLs by at least two complementary activities: a specific effect on genes that define a given cell type by high BRD4 loading at enhancers and the selective suppression of transcription at E2F- and MYC- driven target genes. + Contributed equally Disclosures: Qi: Patent for JQ1: holds patent for JQ1, holds patent for JQ1 Patents & Royalties. Young:Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Enzon Pharmaceuticals: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Bradner:Tensha Therapeutics: Equity Ownership, Scientific founder of Tensha which is translating drug-like derivatives of the JQ1 chemical probe of BET bromodomains used in this study, as cancer therpeutics. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other; Syros Pharmaceuticals: Equity Ownership, Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity., Scientific founder of Syros which is discovering Super Enhancers as a new class of gene control elements. As such, the Dana-Farber Cancer Institute and Dr. Bradner have been granted minority equity. Other.

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