Specific Targeting of KRAS Using a Novel High-Affinity KRAS Antisense Oligonucleotide in Multiple Myeloma
Authors: Sacco Antonio , Federico Cinzia , Todoerti Katia , Ziccheddu Bachisio , Giacomini Arianna , Ravelli Cosetta , Maccarinelli Federica , Bianchi Giada , Belotti Angelo , Ribolla Rossella , Favasuli Vanessa , Revenko Alexey , MacLeod A. Robert , Willis Brandon , Cai Hongoo , Hauser Joana , Rooney Claire , Ambrose Helen , Staniszewska Anna , Hanson Lyndsey , Rossi Giuseppe , Ronca Roberto , Neri Antonino , Mitola Stefania , Bolli Niccolò , Presta Marco , Moschetta Michele , Ross Sarah , Roccaro Aldo M. 
Published: November 2019

INTRODUCTION. The multiple myeloma (MM) mutational landscape has identified KRAS as the most recurring somatic variant, observed in around 26% of cases, therefore KRAS may represent an important therapeutic target. Despite several attempts to develop a targeted therapeutic for KRAS mutant cancers, either direct KRAS enzymatic inhibition, or inhibition of MAPK- and PI3K- downstream effector cascades have not been successful. Therefore, there is a need to develop novel therapeutic approaches that may target the KRAS mutational event in MM. We have studied AZD4785, a novel, potent and selective high affinity 2'-4' constrained ethyl residues containing therapeutic antisense oligonucleotide (ASO) targeting KRAS, both in vitro and in vivo.
METHODS. AZD4785 productive uptake was assessed by measuring KRAS knockdown at both the mRNA and protein level. Molecular mechanisms underlying AZD4785-dependent anti-MM activity were studied, interrogating the transcriptome profiling of AZD4785-treated MM cells. Anti-MM activity of AZD4785 was assessed in vitro in the context of primary MM patients' derived bone marrow stromal cells (BMSCs). Endpoints included evaluation of cell proliferation, cytotoxicity, cell cycle modulation, apoptosis, MM cell migration and adhesion; modulation of MAPK-, PI3K-, apoptotic-signaling. KRAS-mutated (MM1S; KMS20); -wild type (U266; KMS11) MM cell lines; BM MM patients' and peripheral blood healthy donor derived cells were tested. A non-targeting ASO (ASO-ctrl) was used as control. Synergism between AZD4785 and bortezomib, in modulating MM growth was tested. AZD4785-dependent modulation of tumor growth was studied in vivo in a subcutaneous MM.1S.-Luc model and a disseminated GFP/Luc-MM.1S model (BLI); MM cell dissemination to distant BM niches was studied ex vivo, using confocal laser scanning microscopy.
RESULTS. AZD4785 led to specific dose-dependent inhibition of KRAS mRNA and protein expression, in KRAS-mutant, -wild-type cell lines and MM patient-derived CD138+ cells; without affecting NRAS and HRAS content. Wide mRNA transcriptome was performed using AZD4785 treated MM.1S cells vs control: GSEA showed down-regulation of MAPK, cell cycle, TP53 signaling pathways (FDR<0.25; P<0.05) in AZD4785-treated MM cells. Functionally, AZD4785 significantly impaired proliferation and survival of KRAS-mutant MM cells in a dose- and time-dependent manner even in the presence of patients' derived BM-MSCs. Cell growth of KRAS-wild type MM cells was not significantly affected. AZD4785 did not target healthy donors' derived PBMCs. Consistently with the effect on cell growth, AZD4785-treated KRAS mutant MM cells showed S-phase down-regulation, increased of G0/G1 phase and increased apoptotic rate, supported by up-regulation of cleaved-caspase-3, -PARP and BIM. The efficacy of AZD4785 in targeting MM cells within the context of the BM milieu was tested, revealing AZD4785-dependent impairment of MM cell adhesion and migration towards primary BM-MSCs, supported by inhibition of paxillin, cofilin, Src. Protein studies showed inhibition of both MAPK (phospho(p)-ERK1/2, p-MEK, p-RSK90, p-CRAF), and PI3K-Akt signaling pathways, selectively in AZD4785-treated KRAS mutant cells. AZD4785-dependent anti-MM activity was potentiated by the combinatory use of bortezomib, resulting in a significantly higher inhibition of MM cell proliferation, induction of apoptosis, and cell cycle arrest. AZD4785 exerted in vivo down-regulation of KRAS and anti-tumour activity in MM models, being more efficacious when used in combination with bortezomib, in terms of both inhibition of tumor growth and MM cell BM niches colonization, as evaluated by using in vivo whole body-bioluminescence imaging and ex vivo confocal laser scanning microscopy, respectively.
CONCLUSION. Taken together, these data suggest that AZD4785 may represent a novel therapeutic approach for targeting mutant KRAS in MM, either alone or in combination with proteasome inhibitors; and warrant further development.

Disclosures
Giacomini: Fondazione Cariplo: Research Funding. Belotti:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Revenko:Ionis Pharmaceuticals: Employment. MacLeod:Ionis Pharmaceuticals: Employment. Willis:AstraZeneca: Employment. Cai:AstraZeneca: Employment. Hauser:AstraZeneca: Employment. Rooney:AstraZeneca: Employment. Ambrose:AstraZeneca: Employment. Staniszewska:AstraZeneca: Employment. Hanson:AstraZeneca: Employment. Rossi:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. Ronca:Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Bolli:GILEAD: Other: Travel expenses; JANSSEN: Honoraria; CELGENE: Honoraria. Moschetta:AstraZeneca: Employment. Ross:AstraZeneca: Employment. Roccaro:Celgene: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding.