Sajjeev Jagannathan

Pharmacologic screens reveal metformin that suppresses GRP78-dependent autophagy to enhance the anti-myeloma effect of bortezomib

Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.

MicroRNA theragnostics for the clinical management of multiple myeloma

Theragnostics represent cutting-edge, multi-disciplinary strategies that combine diagnostics with therapeutics in order to generate personalized therapies that improve patient outcome. In oncology, the approach is aimed at more accurate diagnosis of cancer, optimization of patient selection to identify those most likely to benefit from a specific therapy and to generate effective therapeutics that enhance patient survival. MicroRNAs (miRNAs) are master regulators of the human genome that orchestrate myriad cellular pathways to control growth during physiologic and pathologic conditions. Compelling evidence shows that miRNA deregulation promotes events linked to tumor initiation, metastasis and drug resistance as seen in multiple myeloma (MM), an invariably fatal hematologic malignancy. miRNAs are readily detected in body fluids, for example, serum, plasma, urine, as well as circulating tumor cells to demonstrate their potential as readily accessible, non-invasive diagnostic and prognostic biomarkers and potential therapeutics. Specific miRNAs are aberrantly expressed early in myelomagenesis and may more readily detect high-risk disease than current methods. Although only recently discovered miRNAs have rapidly advanced from preclinical studies to evaluation in human clinical trials. The development of miRNA theragnostics should provide widely applicable tools for the targeted delivery of personalized medicines to improve the outcome of patients with MM.

MiR-29b replacement inhibits proteasomes and disrupts aggresome+autophagosome formation to enhance the antimyeloma benefit of bortezomib

Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Although proteasome inhibitors (PIs) have transformed management of multiple myeloma (MM), drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism. Genome-wide profiling identified microRNAs (miRs) differentially expressed in bortezomib-resistant myeloma cells compared with drug-naive cells. The effect of individual miRs on proteasomal degradation of short-lived fluorescent reporter proteins was then determined in live cells. MiR-29b was significantly reduced in bortezomib-resistant cells as well as in cells resistant to second-generation PIs carfilzomib and ixazomib. Luciferase reporter assays demonstrated that miR-29b targeted PSME4 that encodes the proteasome activator PA200. Synthetically engineered miR-29b replacements impaired the growth of myeloma cells, patient tumor cells and xenotransplants. MiR-29b replacements also decreased PA200 association with proteasomes, reduced the proteasome’s peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through ubiquitin-independent mechanisms. Immunofluorescence studies revealed that miR-29b replacements enhanced the bortezomib-induced accumulation of ubiquitinated proteins but did not reveal aggresome or autophagosome formation. Taken together, our study identifies miR-29b replacements as the first-in-class miR-based PIs that also disrupt the autophagy pathway and highlight their potential to synergistically enhance the antimyeloma effect of bortezomib.

Pharmacogenomics and chemical library screens reveal a novel SCFSKP2 inhibitor that overcomes Bortezomib resistance in multiple myeloma

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1–Cullin-1–Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.

Genomic integration of Wnt/β-catenin and BMP/Smad1 signaling coordinates foregut and hindgut transcriptional programs

Digestive system development is orchestrated by combinatorial signaling interactions between endoderm and mesoderm, but how these signals are interpreted in the genome is poorly understood. Here we identified the transcriptomes of Xenopus foregut and hindgut progenitors, which are conserved with mammals. Using RNA-seq and ChIP-seq we show that BMP/Smad1 regulates dorsal-ventral gene expression in both the endoderm and mesoderm, whereas Wnt/β-catenin acts as a genome-wide toggle between foregut and hindgut programs. Unexpectedly, β-catenin and Smad1 binding were associated with both transcriptional activation and repression, with Wnt-repressed genes often lacking canonical Tcf DNA binding motifs, suggesting a novel mode of direct repression. Combinatorial Wnt and BMP signaling was mediated by Smad1 and β-catenin co-occupying hundreds of cis-regulatory DNA elements, and by a crosstalk whereby Wnt negatively regulates BMP ligand expression in the foregut. These results extend our understanding of gastrointestinal organogenesis and of how Wnt and BMP might coordinate genomic responses in other contexts. Summary: Genome-wide analysis of Wnt and BMP targets in the Xenopus gut endoderm reveals cooperation and crosstalk between these pathways through integration of Smad1 and β-catenin on cis-regulatory elements.

Abstract 153: Long non-coding RNAs deregulated in Multiple Myeloma impact therapeutic response to proteasome inhibitors

Multiple Myeloma (MM) is a complex disease that frequently leads to fatal outcome and accurate risk classification to optimize the choice of therapy may have impact on clinical outcomes. MM stratification based upon cytogenetic abnormalities and protein-coding gene expression does not adequately correlate with the depth or durability of therapeutic response. Therefore, the new class of molecular effectors, non-coding RNAs (ncRNAs), may enhance the basic understanding of myelomagenesis, drug resistance and provide better stratification of myeloma subtypes. NcRNAs include long ncRNAs (lncRNAs) and microRNAs (miRNAs) that may act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. We hypothesized that lncRNAs commonly deregulated in the 3 resistant cell lines would have significant overlap to the deregulated lncRNA in MM patients. To investigate the role of lncRNAs in resistance to proteasome inhibitors (PIs), we compared global lncRNA profiling in drug-naive cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. RPMI 8226 cells resistant to each of the three PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix 3.0 microarray chips LncRNA expression profiles from drug-resistant cells were compared to that of drug-naive cells treated with vehicle alone. MM patients’ bone marrow aspirates were obtained after UCCOM IRB-approval. Bioinformatic analysis identified a panel of 18 lncRNAs that were significantly (>100-fold) deregulated in all three drug-resistant cell lines relative to drug-naive cells. Strikingly, the majority of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines (Figure 2a). RPMI 8226 cells carry a t(14,16) and none of the deregulated lncRNAs detected localized to chromosomes 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. We also identified lncRNAs deregulated in MM patient samples relative to plasma cells from healthy age-matched individuals. We found a significant overlap (>90%) between deregulated lncRNAs in PI-resistant cells and MM cells. The lncRNA COL4A-2A was upregulated >5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Taken together, we identified a curated panel of deregulated lncRNAs in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors and MM patients. Further investigation is warranted to shed light on the role of these lncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Rebekah Karns, Anil G. Jegga, Sajjeev Jagannathan, Nikhil Vad, Mohamed A.Y Abdel Malek, James J. Driscoll. Long non-coding RNAs deregulated in Multiple Myeloma impact therapeutic response to proteasome inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2015-153

Microbiota-sensitive epigenetic signature predicts inflammation in Crohn’s disease

Altered response to the intestinal microbiota strongly associates with inflammatory bowel disease (IBD); however, how commensal microbial cues are integrated by the host during the pathogenesis of IBD is not understood. Epigenetics represents a potential mechanism that could enable intestinal microbes to modulate transcriptional output during the development of IBD. Here, we reveal a histone methylation signature of intestinal epithelial cells isolated from the terminal ilea of newly diagnosed pediatric IBD patients. Genes characterized by significant alterations in histone H3-lysine 4 trimethylation (H3K4me3) showed differential enrichment in pathways involving immunoregulation, cell survival and signaling, and metabolism. Interestingly, a large subset of these genes was epigenetically regulated by microbiota in mice and several microbiota-sensitive epigenetic targets demonstrated altered expression in IBD patients. Remarkably though, a substantial proportion of these genes exhibited H3K4me3 levels that correlated with the severity of intestinal inflammation in IBD, despite lacking significant differential expression. Collectively, these data uncover a previously unrecognized epigenetic profile of IBD that can be primed by commensal microbes and indicate sensitive targets in the epithelium that may underlie how microbiota predispose to subsequent intestinal inflammation and disease.

Abstract A18: MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib.

Purpose: The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin+proteasome system (UPS) and also disrupt the aggresome+autophagy pathway for anti-myeloma benefit. Background: Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches. Drug resistance accounts for the majority of tumor relapses and cancer-related deaths. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome+autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Experimental Procedures: Myeloma cells were exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naive cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly reduced in drug-resistant cells. To determine the effect of individual miRNAs, synthetic miRNA replacements were transfected into myeloma cells. Individual miRNA replacements were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient tumor cells relative to plasma cells form healthy individuals. MiRNA-29b targeted PSME4 which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4 and the protein product PA200, a proteasome activator. Reduction in PA200 reduced the proteasome9s chymotrypsin-like peptidase activity and inhibited ornithine decarboxylase turnover, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble ubiquitinated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. The ubiquitinated proteins also were not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and uncouple the UPS from the aggresome-autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma effect of PIs. Citation Format: Sajjeev Jagannathan, Nikhil Vad, Mohamed Abdel-Malek, Ehsan Malek, James Driscoll. MicroRNA-29b replacement inhibits proteasomes and disrupts the aggresome-autophagy pathway to enhance the antimyeloma benefit of bortezomib. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A18.

Abstract LB-140: The Hsp90 inhibitor ganetespib overcomes EGFR-based intratumoral heterogeneity to block glioma proliferation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Intratumoral heterogeneity is evident as distinct subpopulations of cancer cells within and between tumor lesions that profoundly impact therapeutic response and patient outcome. While the advent of targeted therapies to exploit a tumors dependence on critical proliferative or survival pathways has significantly improved patient outcomes in a range of solid tumor types, it is also apparent that targeted therapeutics do not help all molecularly selected patients and even when clinical benefit is observed, it is often of limited duration. To address this question, we have explored the invariably fatal adult brain tumor glioblastoma multiforme (GBM). Heterogeneous forms of the epidermal growth factor receptor (EGFR) arise through amplification or mutation as signature pathogenetic events in this highly aggressive disease. While EGFR deregulation promotes tumor cell proliferation, drug resistance and tumor survival, functional blockade of the EGFR kinase activity alone does not achieve maximal therapeutic benefit. Since intratumoral heterogeneity represents a major impediment to the development of effective therapeutics in GBM, we sought to identify small molecules cytotoxic to glioma cells engineered to overexpress either wildtype (WT), constitutively active mutant EGFR-vIII, kinase-dead (KD) form or a basal level of EGFR-WT using a live cell-based, high-throughput screen (HTS) of ∼2,000 FDA-approved or bioactive compounds. Our screen revealed that the Hsp90 inhibitor ganetespib (STA-9090, Synta Pharmaceuticals, Lexington, MA) was highly cytotoxic (IC50 ∼20nM) to glioma cells that overexpressed each of the EGFR forms. Real-time measurements were then performed to demonstrate that ganetespib at nM concentrations reduced the proliferation of brain tumor cells expressing EGFR-WT, -vIII or KD forms, induced apoptosis and inhibited glioma migration. Ganetespib also potently reduced the proliferation of human GBM patient-derived tumor-initiating stem cells but not that of untransformed glial cells. Ganetespib effectively impaired the growth of subcutaneously implanted human brain tumor xenotransplants that expressed either EGFR-WT, -vIII or both EGFR forms placed in immunocompromised mice. In addition, ganetespib provided a survival benefit compared to untreated mice. The Hsp90 inhibitor ganetespib is a highly potent agent that impairs the growth-promoting functional kinase-dependent and independent activities of EGFR to overcome context-dependent intratumoral heterogeneity with potential benefits for GBM patients. Citation Format: James J. Driscoll, Nitin S. Narayanan, Sajjeev Jagannathan. The Hsp90 inhibitor ganetespib overcomes EGFR-based intratumoral heterogeneity to block glioma proliferation. [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 LB-140. doi:10.1158/1538-7445.AM2014-LB-140

Abstract 1462: Tandem genome-wide and functional screening reveals that MiRNA-29 regulates the proteasome activator PSME4 to promote therapeutic resistance in myeloma

MicroRNAs (miRNAs) are newly recognized, master regulators of the human genome and hence play key roles in cellular pathways that govern proliferation, differentiation and apoptosis during both physiologic and pathologic states. Accordingly, alterations in miRNA levels contribute to myriad diseases including human cancers. MiRNAs have rapidly attracted significant attention as agents with significant potential as diagnostics and therapeutics to improve the outcome of cancer patients. While proteasome inhibitors (PIs) such as bortezomib (Velcade, Millennium-Takeda) have transformed management of the invariably fatal disease multiple myeloma, therapeutic resistance inevitably emerges through mechanisms that remain elusive. A goal of these studies was to investigate the role of individual miRNAs in regulating functional activity of the ubiquitin+proteasome system. Genome-wide expression profiling was performed to identify differentially expressed miRNAs in the PI-resistant cells relative to drug-naive parental cells. A live-cell functional screen that employed a recombinant fluorescent probe comprised of a short-lived reporter bearing a destabilizing modification to promote constitutive proteasomal degradation was used to quantitate the effect of individual miRNAs. MiRNAs-29a/b/c were significantly reduced in the PI-resistant cells and these miRNAs negatively regulated PSME4 which expresses the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29 replacements reduced PSME4 levels, promoted myeloma cell death and overcame drug resistance. Deletion of a nuclear localization signal (NLS) enriched the cytoplasmic distribution and enhanced the functional effect of miRNA-29 replacements. Doxycycline-induced expression of miRNA-29b or miRNA-29 variants that lacked the NLS also dramatically reduced the in vivo growth of subcutaneously injected myeloma tumor cells in NOD/SCID mice. We demonstrate that the loss of a tumor suppressive miRNA activates not only inherently oncogenic pathways to promote the generation of a cancer phenotype but also to promote therapeutic resistance to PIs. We provide a platform to perform a genome-wide interrogation of miRNAs and other non-coding RNAs that regulate the ubiquitin+proteasome system. The significant cytotoxic effect of miRNA-29 replacements observed using MM cell lines and myeloma patient samples as well as ability to reduce tumor growth in vivo validate miRNA-29 replacements as highly effective anti-cancer tools to overcome therapeutic resistance, promote tumor cell death and eventually improve patient outcome. Citation Format: James J. Driscoll, Sajjeev Jagannathan. Tandem genome-wide and functional screening reveals that MiRNA-29 regulates the proteasome activator PSME4 to promote therapeutic resistance in myeloma. [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 1462. doi:10.1158/1538-7445.AM2014-1462