p62-Dependent Autophagic Reserve Maintains Protein Homeostasis and Determines Proteasome Inhibitor Susceptibility in Multiple Myeloma Cells

Abstracts are ongoing to inform our efforts of clinically translating CAR T-cell therapy in this entity.

BP-037 Role of RECQ1 helicase in multiple myeloma drug resistance E. Viziteu,1 B. Klein,1,2,3 J. Basbous,1 Y.L. Lin,1 A. Seckinger,4 C. Gourzones,1 C. Grandmougin,1 A. Constantinou,1 H. Goldschmidt,4 P. Pasero,1 D. Hose,4 J. Moreaux1,2,3 1

Institute of Human Genetics, CNRS UPR 1142, Montpellier, France;

2

Laboratory for Monitoring Innovative Therapies, Department of Bio-

logical Hematology, CHRU Montpellier, France; 3Montpellier University, UFR Medecine, Montpellier, France; 4Medizinische Klinik V, Universitätsklinikum Heidelberg and Nationales Centrum für Tumorerkrankungen, Heidelberg, Germany

Using a microarray-based genome-wide screen for genes responding to DNA methyltransferases (DNMT) inhibition in MM cells, we identified RECQ1 among the genes downregulated by DNMT inhibitor. RECQ helicase are DNA unwinding enzymes involved in the maintenance of chromosome stability. RECQ1 silencing in cancer cells results in mitotic catastrophe and prevents tumor growth in murine models. RECQ1 is significantly overexpressed in primary myeloma cells compared to normal plasma cells and in myeloma cell lines compared to primary myeloma cells of patients. High RECQ1 expression is associated with a poor prognosis in two independent cohorts of patients. RECQ1 knock down inhibits growth of myeloma cells and induces apoptosis. Given the known role of RECQ1 in replication and DNA repair activation, the effect of RECQ1 depletion in DNA damage response was investigated. RECQ1 depletion induced spontaneous accumulation of DNA double strand breaks (DSBs) evidenced by the phosphorylation of ATM and H2AX histone and detection of 53BP1 foci. Using an alkaline comet assay, a significant increase in DNA strand breaks was confirmed in RECQ1 depleted cell lines compared to control. RECQ1 depletion was associated with CHK1 and CHK2 phosphorylation in MM cells. Since RECQ1 depletion is associated with DNA damage response activation and DNA strand breaks formation, a link between RECQ1 expression and drug sensitivity was hypothesized. RECQ1 overexpression significantly protects myeloma cell lines from melphalan and bortezomib-induced apoptosis. Furthermore, RECQ1 depletion sensitizes myeloma cells to treatment. Using immunoprecipitation, RECQ1 was shown to interact with PARP1 but not RAD51 or MSH2. An increased association of the two proteins was found upon DNA damages induced by melphalan. In agreement, RECQ1 depletion sensitizes myeloma cell lines to PARP inhibitor. We identified RECQ1 as a miR-203 target. Interestingly, aberrant methylation of miR-203 was reported in MM cells and treatment with 5-aza-2’-deoxycitidine led to promoter demethylation and miR-203 re-expression. Furthermore, anti-miR-203 treatment induced a significant increase of RECQ1 mRNA level in MM cells.

In conclusion, RECQ1 represent a biomarker of drug resistance in MM, which is targeted by DNMT inhibitors. This suggests association of alkylating agents and/or PARP inhibitors with DNMT inhibitor may represent a therapeutic approach in RECQ1-high patients associated with a poor prognosis.

BP-038 A Plastic SQSTM1/p62-Dependent Autophagic Reserve Maintains Protein Homeostasis and Determines Proteasome Inhibitor Susceptibility in Multiple Myeloma Cells T. Perini,1,2,3 E. Milan,1,2 M. Resnati,1 U. Orfanelli,1 L. Oliva,1 A. Raimondi,4 P. Cascio,5 A. Bachi,6 M. Marcatti,3 F. Ciceri,3 S. Cenci1,2 1

San Raffaele Scientific Institute, Division of Genetics and Cell Biology,

Milano, Italy; 2Università Vita-Salute San Raffaele, Milano, Italy; 3San Raffaele Scientific Institute, Department of Oncohematology, Hematology and Bone Marrow Transplantation Unit, Milano, Italy; 4San Raffaele Scientific Institute, Imaging Research Center, Milano, Italy; 5

Department of Veterinary Sciences, University of Torino, Italy; 6IFOM,

FIRC Institute of Molecular Oncology, Milano, Italy

Background: Multiple myeloma (MM) is the paradigmatic proteasome inhibitor (PI) responsive cancer, but many patients fail to respond. A possible mechanism of primary resistance and an attractive target to enhance sensitivity is (macro)autophagy, the main alternative catabolic pathway, which we recently found essential to bone marrow plasma cells, the normal counterpart of MM. Methods: Here, using MM cell lines and primary cells, we integrated comparative proteomics with electron microscopy, fluorescent imaging, lentiviral engineering, and molecular biology techniques to address the role of autophagy in the maintenance of protein and organelle homeostasis in MM cells, and the underlying mechanisms. Results: We found that in MM cells abundant autophagy alleviates proteasomal load by constitutively disposing of substantial amounts of ubiquitinated proteins and by selectively targeting the endoplasmic reticulum (ER), a chief source of proteasome substrates, disclosing a co-operation between the ubiquitinproteasome system (UPS) and autophagy closer than previously thought. Indeed, autophagy inhibition or stimulation greatly sensitizes to, or protects from, PI-induced protein aggregation and cell death. Moreover, we found such co-operation to depend on the autophagic cargo receptor/adapter protein SQSTM1/p62. Lentiviral engineering revealed that p62 is essential for MM cell survival and affords specific PI resistance. Under PIs, myeloma cells selectively enhance p62 de novo expression and reset its vast endogenous interactome, diverting p62 from signaling partners to maximize its association with ubiquitinated proteins for autophagic degradation. Of clinical relevance, saturation of such autophagic reserve, as indicated by intracellular accumulation of undigested p62+ aggregates, neatly discriminates patient-derived myelomas inherently susceptible to PIs from primarily resistant ones. Conclusions: We disclosed an essential role for autophagy in protein and organelle

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Abstracts homeostasis in MM cells. In particular, we identified p62 as an essential component of an autophagic reserve that not only synergizes with the UPS to maintain proteostasis, but also mediates a plastic adaptive response to PIs. Altogether, these data integrate autophagy into our previously established proteasome load-versuscapacity model of PI responsiveness, and reveal p62 aggregation as a faithful marker of defective proteostasis, defining a novel prognostic and therapeutic framework for MM.

BP-039 TOP2A knockdown resensitizes carfilzomib-resistant HMCLs to carfilzomib A. Reale, T. Khong, S. Mithraprabhu, M. Ramachandran, I. Savvidou, A. Spencer Antonia Reale: Myeloma Research Group, Australian Centre for Blood Diseases, The Alfred Hospital/Monash University, Melbourne,

factor for patient selection for specific protocols or as independent prognostic marker in solid tumors. TOP2A was also overexpressed in the ‘proliferation cluster’ associated with greater proliferation rate and poor outcome in newly diagnosed MM patients. Suppression of TOP2A by siRNA in carfilzomib-resistant HMCLs significantly resensitised the cell lines to carfilzomib. Conclusion: Our results suggest that TOP2A is overexpressed in carfilzomib-resistant HMCLs indicating a possible role as a predictive marker of response to carfilzomib in MM.

BP-040 IMiDs induced Ikaros-dependent downregulation of MYC in myeloma is mediated through the repression of the 3’ IGH enhancer activity and the NURD complex repositioning

Australia; School of Medical Oncology, Department of Biological

P. Neri, I. Tagoug, J. Simms, R. Maity, V. Zepeda, P. Duggan, N.J. Bahlis

Sciences and Human Oncology, University of Bari ‘Aldo Moro’, Italy;

Southern Alberta Cancer Research Institute, University of Calgary

Tiffany Khong, Sridurga Mithraprabhu, Malarmathy Ramachandran, Ioanna Savvidou:Myeloma Research Group, Australian Centre for Blood Diseases, The Alfred Hospital/Monash University, Melbourne, Australia; Andrew Spencer: Myeloma Research Group, Australian Centre for Blood Diseases, The Alfred Hospital/Monash University, Melbourne, Australia; Malignant Hematology and Stem Cell Transplantation, The Alfred Hospital, Melbourne, Australia and Department of Clinical Hematology, Monash University, Clayton, Australia

Introduction/Background: Multiple myeloma (MM) remains incurable despite the introduction of novel therapeutic agents. Microarray-based technologies were adopted in our study to determine if a genetic signature associated with resistance to carfilzomib, a second-generation proteasome inhibitor already in use in clinical settings, could be identified. Materials and Methods: 18 genetically heterogeneous human myeloma cell lines (HMCLs) were treated with carfilzomib and a cell viability profile was assessed categorizing the HMCLs as sensitive, intermediate or resistant to carfilzomib. Following categorization gene expression profiling was performed and validated with q-RT-PCR and knockdown assays. Results: 29 genes were differentially regulated between the sensitive and resistant cell lines. Top genes based on intensity values and biological significance were: LOC731314, TSPAN13, APH1B, TSPYL5, COX7B2, PCSK1N, LRRC38, TCIRG1, TOP2A, ADM2, ITM2A, TSPAN13, STOM, UBE2C, SNHG8. Gene ontology (GO) enrichment analysis identified two pathways that were significantly different between the resistant and sensitive HMCLs; pathogenic escherichia coli infection (p¼0.002) and lysosome (p¼0.006). Eight GO terms were enriched: 4 related to biological processes and 4 related to cellular components. TOP2A, an enzyme that controls and alters the topologic states of DNA during transcription and is involved in cell cycle and proliferation, was identified to be overexpressed in resistant HMCLs. It functions as the target for several anticancer agents and a variety of mutations in this gene have been associated with the development of drug resistance. TOP2A may be used as a predictive

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Clinical Lymphoma, Myeloma & Leukemia September 2015

MYC rearrangement is a recurrent somatic event in MM and results in MYC repositioning near superenhancers such as the 3’ IGH enhancers. IMiDs promote the proteasomal degradation of IKZF1/3 and the transcriptional repression of MYC. However the mechanism through which IMiDs downregulate MYC is not well understood. Transcriptome analysis (RNAseq) of MM cells treated with lenalidomide or upon silencing of IKZF1 confirmed the downregulation of MYC, IRF4 and among others the upregulation of CD38, DKK1, PDL1 and a host of genes involved in type I & II interferon response. Gene set enrichment analysis (GSEA) confirmed the enrichment of genes signatures associated with MYC as well as the interferon response. Notably, in t(4;14) MM cell lines we observed a significant downregulation of FGFR3 while the expression of WHSC1 was not affected. As FGFR3 is driven by the 3’ IGH enhancer while WHSC1 is under the control of the intronic E enhancer, and in view of the known role of IKZF1 in CSR, we speculated that IKZF1 regulates the 3’ enhancer activity and MYC expression in cells harbouring a MYC:IGH rearrangement. To validate this hypothesis we established the genome-wide distribution of IKZF1 in OPM2 cells by ChIP-Seq. Ikaros bound to 17660 loci of which 43% were associated with gene targets. Ikaros-binding motifs and other motifs such as STAT1, E2A, RUNX1 were also identified (MEME Tomtom and Jasper motif analysis) in the vicinity of Ikaros enrichment peaks. Importantly Ikaros peaks also mapped to the IGH 3’ enhancer loci and these results were confirmed by ChIP-PCR analysis. Of interest while no Ikaros peaks mapped to MYC (within 5 kb of TSS) by ChIP-seq, modest enrichment of Ikaros at MYC promoter was identified by ChIP-PCR in MM1S and KMS11 cells, and this enrichment was significantly attenuated by lenalidomide treatment. Lastly and since Ikaros is a known integral component of the NuRD (nucleosome remodelling and histone deacetylase) complex, we examined whether lenalidomide treatment and Ikaros depletion lead to local gain of NuRD at MYC TSS. CHD4 ChIP-PCR analysis revealed