// <![CDATA[ $('.header-date').hide();$('#titleAuthors').hide();$('#abstractHeader').hide(); // ]]> William Bruno1, Claudia Martinuzzi1, Virginia Andreotti1, Lorenza Pastorino1, Francesco Spagnolo2, Bruna Dalmasso1, Francesco Cabiddu3, Marina Gualco3, Alberto Ballestrero1, Giovanna Bianchi-Scarrà1, Paola Queirolo2, Federica Grillo4, *, Luca Mastracci4, *, Paola Ghiorzo1, * on behalf of the Italian Melanoma Intergroup (IMI) 1Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy 2Department of Medical Oncology, IRCCS AOU San Martino-IST, Genoa, Italy 3Department of Pathology, IRCCS AOU San Martino-IST, Genoa, Italy 4Department of Surgical and Diagnostic Sciences, Pathology Unit, University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy *These authors have contributed equally to this work Correspondence to: Martinuzzi Claudia, email: claudia.martinuzzi@edu.unige.it Keywords: BRAF, primary melanoma, PNA-clamping, immunohistochemistry, heterogeneity Abbreviations: PNA: peptide nucleic acid – PNA-clamping real-time PCR; IHC: immunohistochemistry; NGS: next generation sequencing; FFPE: formalin-fixed, paraffin embedded Received: September 20, 2016 Accepted: November 24, 2016 Published: December 22, 2016

Metabolic reprogramming is emerging as a cancer vulnerability that could be therapeutically exploitable using different approaches, including amino acid depletion for those tumors that rely on exogenous amino acids for their maintenance. ʟ-Asparaginase (ASNase) has contributed to a significant improvement in acute lymphoblastic leukemia outcomes; however, toxicity and resistance limit its clinical use in other tumors. Here, we report that, in multiple myeloma (MM) cells, the DNA methylation status is significantly associated with reduced expression of ASNase-related gene signatures, thus suggesting ASNase sensitivity for this tumor. Therefore, we tested the effects of ASNase purified from Erwinia chrysanthemi (Erw-ASNase), combined with the next-generation proteasome inhibitor (PI) carfilzomib. We observed an impressive synergistic effect on MM cells, whereas normal peripheral blood mononuclear cells were not affected. Importantly, this effect was associated with increased reactive oxygen species (ROS) generation, compounded mitochondrial damage, and Nrf2 upregulation, regardless of the c-Myc oncogenic-specific program. Furthermore, the cotreatment resulted in genomic instability and DNA repair mechanism impairment via increased mitochondrial oxidative stress, which further enhanced its antitumor activity. Interestingly, carfilzomib-resistant cells were found to be highly dependent on amino acid starvation, as reflected by their higher sensitivity to Erw-ASNase treatment compared with isogenic cells. Overall, by affecting several cellular programs, Erw-ASNase makes MM cells more vulnerable to carfilzomib, providing proof of concept for clinical use of this combination as a novel strategy to enhance PI sensitivity in MM patients.

Cancer cells fuel growth and energy demands by increasing their NAD⁺ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD⁺-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD⁺ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD⁺-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD⁺-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.