The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.

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Multiple Myeloma (MM) is a clonal B-cell malignancy characterized by accumulation of malignant plasma cells (PCs) within the bone marrow (BM) in close contact with stromal cells (SCs) which secrete growth factors and cytokines, promoting tumor cell growth and survival. The rapid progression of MM is dependent upon cellular interactions within the BM microenvironment, and novel agents targeting this interaction appear to be promising therapeutic strategies for the treatment of MM tumor expansion. Unlike most other organs, the BM microenvironment is physiologically hypoxic, a pre-requisite for normal BM hematopoiesis. It is well established that hypoxia is an important selective force in the evolution of tumor cells and a stabilization of HIF-1α protein has been documented in several human cancers. While the role of hypoxia in the pathogenesis of hematologic malignancies has yet to be elucidated, recent animal studies have shown that changes in oxygen levels within the BM microenvironment support the survival and expansion of MM cells. Furthermore, some drugs active in MM, such as Bortezomib and Lenalidomide, are believed to exert their effects in part by interfering with hypoxia-induced signaling cascades. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of HIF-1α in the PCs-BMSCs interplay. To test this hypothesis, we used EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. We have already shown that EZN-2968 is highly specific for HIF-1α mRNA and it results in a long lasting and time dependent inhibition of HIF-1α protein level. Herein, we provide evidence that the interaction between MM cells and BMSCs is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BMSCs were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed that EZN-2968 down-modulates cytokine-induced signaling cascades after a short incubation, and seems to induce a negative modulation of those transcripts previously shown to reflect the activation state of specific tumor cell pathways (cell proliferation and survival). This observation was also supported by gene expression profile experiments. One of the key finding of our study is that PC attachment to the extracellular matrix protein was markedly reduced in the presence of EZN-2968. The effects of HIF inhibition on MM cell adhesion are quite intriguing, since MM pathogenesis is dependent upon the interaction of MM cells with the SCs. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between BMSCs and PCs in MM. We conclude that HIF inhibition may be an attractive therapeutic target for MM. Citation Format: Enrica Borsi, Giulia Perrone, Carolina Terragna, Marina Martello, Angela Flores Dico, Lucia Pantani, Annamaria Brioli, Giovanni Martinelli, Michele Cavo. HIF-1α inhibition blocks the cross talk between multiple myeloma plasmacells and tumour microenvironment. [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 3608. doi:10.1158/1538-7445.AM2014-3608

Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics. Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence. Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively. Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed. SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A). Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (<-2FC>2; FDR=0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed in every analyzed cell compartment; on the contrary, Hedgehog pathway was overall down regulated. Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells. Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply. Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Intro: Multiple Myeloma (MM) is a biologically complex disease, whose genetic plasticity favors the coexistence of genetically heterogeneous subclones, selected in a Darwinian fashion throughout the disease course. Therapy might represent a major selective pressure over the different subclones, thus supporting an evolutionary model of the disease. Aim: To explore the existence of different clonal evolution patterns in MM, eventually driven by therapeutic selective pressure. P&M The study included 33 pts with symptomatic MM, up-front treated either with combination regimens including a proteasome inhibitor (28), or with cyclophosphamide. For each pts, paired BM samples were collected both at diagnosis and at relapse. SNPs array analyses were performed on the CD138+ enriched cell fractions. Results: Two approaches were applied: a) monitoring the variations of macro CNAs; b) focusing on changes of CNAs frequencies, as observed in 27 genes of interest. Both approaches were consistent in highlighting three major evolution patterns: in 7/33 (21%) pts, the genomic background at relapse was almost identical to that of diagnosis. In 13/33 (39%) pts, an overall increase in the frequencies of the same CNAs as observed at diagnosis was detected at relapse. Finally, in 13/33 (39%) pts, either increased or decreased frequencies of several CNAs, as well as several differences in the CNAs type’s prevalence were observed at relapse, as compared to diagnosis. Of interest, even if an overall CNAs median number increase was observed from diagnosis to relapse (226 vs 507, respectively) - supported by acquisition of CNAs either commonly described as secondary genomic events (i.e. del17p13, amp1q21, del1p23), or associated to the resistance to bortezomib (i.e. del8p21) - any peculiar CNAs resulted significantly prevalent in the 3 identified subgroups of pts. A high rate (92%) of achievement of VGPR or better quality of response to upfront therapy characterized the third subgroup of pts, whereas the rate of VGPR in the remaining pts was only 20% and PR or SD were observed in 9 and 7 pts, respectively. Finally, the median time to first progression of this subgroup of pts was significantly shorter as compared to that of pts with branching evolution (24 vs 35 months, range 4-41 and 7-123 months, respectively, p=0,01). Conclusion: The genomic architecture of a subgroup of relapsed MM pts, up-front responsive to new drugs-based combination therapies, resulted overall different from that of diagnosis, suggesting a branching evolution of the disease, sustained by the shrinking of the most prevalent clone (therapy-sensitive), as well as by the expansion of subclones (therapy-resistant) not already evident at diagnosis. This observation raises the question whether re-treatment of relapsed pts should be appropriate in the case of branching evolution. Acknowledgements: AIRC (MC), Fondazione Berlucchi (CT), FUV (EB). Citation Format: Carolina Terragna, Marina Martello, Barbara Santacroce, Vincenza Solli, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Beatrice Zannetti, Katia Mancuso, Giulia Marzocchi, Nicoletta Testoni, Gaia Ameli, Rosalinda Termini, Angela Flores Dico, Enrica Borsi, Giovanni Martinelli, Michele Cavo. A branching evolution model at relapse characterizes multiple myeloma patients who responded to upfront combination therapy including new drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3936. doi:10.1158/1538-7445.AM2017-3936