Mohamed A.Y. Abdel Malek

Rare extraperitoneal involvement with fatal outcome in a case of bilateral luteinized thecoma of the ovaries with sclerosing peritonitis.

We report the case of a woman diagnosed with bilateral luteinized thecoma of the ovaries with sclerosing peritonitis, multiple intraperitoneal cystic lesions, and extraperitoneal lesions of the liver, inferior to the spleen, and high suspicion of bone marrow involvement. The patient developed profound pancytopenia with rapid clinical deterioration and a fatal outcome.

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

Abstract LB-245: Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The molecular events that dictate the initiation, progression and therapeutic response in multiple myeloma (MM) remain incompletely understood. The ubiquitin (Ub) ligase Skp1-Cullin-1-Skp2 (SCFSkp2) complex promotes proliferation by inducing proteasomal degradation of the cyclin-dependent kinase inhibitor p27Kip1. Skp2 overexpression and reduced p27 levels are frequent events in human cancers and are associated with poor prognosis. Methods: Prospective pharmacogenomics was performed using next generation exome sequencing gene expression profiles (GEPs) compiled from the APEX/SUMMIT phase III clinical trial deposited in dataset GSE9782. 255 relapsed MM patients were randomized to bortezomib (BTZ) vs. dexamethasone (DEX) alone and progression-free (PFS) and overall survival (OS) then correlated with the pre-treatment GEPs. RPMI8226 cells were exposed for 8 months to successively escalated doses of the proteasome inhibitors BTZ, carfilzomib or ixazomib to generate chemoresistant cell lines. Side population (SP) cells were isolated from MMCLs and patients’ samples by flow cytometry sorting based upon Hoechst 33342 staining. Results: Pharmacogenomic correlation of GEPs with clinical response revealed hyperexpression of CUL1 and SKP2 in tumors from MM patients that did not respond to BTZ. CUL1 and SKP2 hyperexpression was correlated with significantly reduced PFS and OS after treatment with BTZ but not with DEX. Cullin-1 and Skp2 were elevated in cells generated with acquired PI-resistance. CUL1 or SKP2 genetic ablation significantly enhanced the sensitivity of chemoresistant cells to PIs. A high-throughput assay was then employed using GFP-tagged p27 to screen large databases and chemical libraries in order to identify novel lead compounds that inhibited the Skp2-dependent ubiquitination of p27. Treatment with DT204, a novel lead compound identified here, reduced p27 ubiquitination, its accumulation in myeloma cells and impaired Skp2 association with Cullin-1 and Cks1, a critical rate-limiting effector of Skp2 activity. The anti-myeloma effect of DT204 was diminished using a phosphorylation-defective p27 mutant. Co-treatment with DT204 enhanced the anti-myeloma effect of BTZ against MM cell lines, patient tumor cells, stem cells and the tumor-initiating clonogenic side population (SP). The in-vivo efficacy and survival benefit of DT204 as monotherapy or in combination with BTZ will be reported. Conclusion: Taken together, the results demonstrate that pharmacologics to selectively disrupt the SCFSkp2 complex-p27 axis within the UPS holds promise to overcome chemoresistance with therapeutic benefit for myeloma patients. Skp2, Cullin1 can be utilized as biomarkers to select MM patients who will benefit from early experimental therapeutics of Skp2-inhibitors. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Mohamed Abdel Malek, James J. Driscoll. Systems-based pharmacogenomics reveals that the ubiquitin ligase SCF-SKP2 is a molecular determinant of clinical response to bortezomib-based therapy. [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 LB-245. doi:10.1158/1538-7445.AM2015-LB-245

Chapter 2 – Molecular Cross-Talk between the Autophagy and Apoptotic Networks in Cancer

Autophagy (self-eating) and apoptosis (self-killing) are evolutionarily conserved, catabolic pathways that are essential to maintain cellular and organismal homeostasis. The functional and molecular relationship between autophagy and apoptosis is highly complex since, under certain cellular circumstances, autophagy constitutes a stress adaptation to prevent or suppress apoptosis and hence cell death, whereas in other settings, autophagy constitutes an alternative pathway to promote cell death. Autophagy and apoptosis are both triggered by common upstream signals to result in a synergistic effect that stimulates these processes, or in other instances the cell switches between the two responses in a mutually exclusive manner. Therefore, the precise events and molecular effectors that regulate the complex interplay between autophagy and apoptosis as determinants of cell fate under physiologic and pathologic conditions remain poorly understood. Autophagy and apoptosis have now been shown to be connected through several nodes that display rapid, extensive cross-talk that enables their coordinated regulation. Autophagy fulfills an essential role as it facilitates the degradation of potentially oncogenic molecules, preventing the development of cancers, while apoptosis prevents the survival of cancer cells. However, mutation, gene inactivation, and other abnormalities can lead to defective autophagy and/or apoptosis and ultimately the initiation and progression of cancers. Autophagy appears to have a dual role in cancer, as it has now been shown to facilitate the survival of tumor cells in stress conditions such as hypoxic or low-nutrition environments. A more thorough and comprehensive understanding of autophagy and apoptosis is necessary to facilitate the identification of novel drug targets and to develop more effective cancer therapeutics that improve patient survival.

Targeting the Proteasome Pathway for the Treatment of Solid Tumors

The ubiquitin-proteasome system (UPS) is a highly complex protein network that maintains proteostasis and cell viability through the targeted and timely turnover of selected substrates. The proteasome serves as the catalytic core of the UPS to precisely recognize and efficiently execute the rapid ATP-dependent removal of ubiquitinated proteins. Small-molecule pharmacologic inhibitors exploit the pivotal role of the proteasome in cellular metabolism as a molecular vulnerability in cancer cells to promote the selective cytotoxicity of tumor cells. Proteasome inhibitors (PIs) have yielded durable clinically responses that dramatically improve the survival of patients diagnosed with the invariably fatal hematologic malignancy multiple myeloma (MM). Success of the PI bortezomib in the treatment of the hematologic malignancy MM has emerged as the standard of care and catapulted the UPS into a position of prominence as a model system in cancer biology and drug development. However, advancement of PIs to improve the treatment of patients with solid tumors has been far more challenging and less successful. Clinical assessment of second-generation PIs progresses as well as pharmacologics to intervene at other points within the UPS is being explored for both hematologic and solid tumors. Agents to target non-proteolytic activities associated with the proteasome are emerging as are agents to inhibit Ub-binding proteins. New approaches to unravel the UPS should advance its utilization as a drug development platform in mechanism-based anticancer strategies that include PIs as monotherapy or in synergistic combinations that improve the outcome of patients with solid tumors.