Abstract: Abstract 2572 Backgroud: In Japan, bortezomib (Velcade; Janssen Pharmaceutical Corporation, Tokyo, Japan) was approved on October 2006 for relapsed or refractory multiple myeloma, and sales began on the open market in December 2006. The approval process clarified potential risk for interstitial lung disease (ILD) among bortezomib-treated Japanese patients as we reported before (Blood 2006; 107:3492–4, Lancet Oncol 2008; 9:414–5). By 2007, various safety alerts were widely disseminated through the responsible pharmaceutical company, the media, the academic societies and the regulatory authority of Japan (Pharmaceuticals and Medical Devices Agency, PMDA) concerning the potential ILD risk of bortezomib (J Clin Oncol 2008; 26:5820–3). In this study, we investigated whether notification of patients and providers about these safety alerts influenced the clinical outcomes after bortezomib therapy in Japan. Methods: We reviewed all bortezomib-related adverse drug reaction (ADR) reports available from open source database of PMDA from December 2006 to December 2009. The indication of bortezomib remained unchanged in this period. We compared the number of reported cases and their outcomes between the early postmarketing period (from December 2006 to December 2007) and the late period after the dissemination of the safety alerts concerning ILD (from January 2008 to December 2009). Results: In the study period, 419 ADR cases were identified after bortezomib therapy. The proportion of ILD to any ADR was 25.0% (9 of the 36 cases) in December 2006, 8.3% (13 of the 156 cases) in 2007, 12.0% (17 of the 142 cases) in 2008 and 7.1% (6 of the 85 cases) in 2009 (Table 1). When we compared the early postmarketing period before the safety alerts and the late period after the safety alerts, the death proportion of the cases with any ADRs seemed similar. Forty of the 192 cases (20.8%) died in the early period and 47 of the 227 cases (20.7%) in the late period. On the other hand, the death proportion among ILD cases appeared to decrease remarkably (Table 2). Nine of the 22 ILD cases (40.9%) died in the early period and 1 of the 23 ILD cases (4.3%) died in the late period (p=0.004, Fisher's exact test). Conclusions: Notification of patients and providers about the safety alerts appeared to influence the clinical practice and to improve proportion of fatal outcomes for bortezomib-treated ILD patients. This study illusrates the importance of communicating drug safety information to the public. Disclosure: Oshima: Sanofi-Aventis: Employment.

Abstract: Genetic mutations in FLT3 (fms-like tyrosine kinase-3) play an important role in the pathogenesis of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) occur in approximately 25% of all AML cases and various tyrosine kinase inhibitors (TKIs) targeting FLT3-ITD such as quizartinib, crenolanib, and gilteritinib have been developed. Although these selective FLT3 inhibitors were thought to be promising, their effects turned out to be temporary due to the rapid development of resistance associated with clonal switching. Acquired FLT3 point mutations at D835 in the activation loop of tyrosine kinase domain are often accountable for clonal switching at least for Type II TKIs. In addition, adjunct mutations in other genes are also found to be associated with TKI resistance. To investigate the underlying molecular mechanism of this secondary, mutation-driven acquired resistance, we first analyzed co-occurring mutations in the leukemia cells obtained from 26 AML patients with FLT3-ITD (n=14) or FLT3-ITD/D835 dual (n=12) mutations, and performed cap analysis of gene expression (CAGE) sequencing, which identifies and quantifies the 5' ends of capped mRNA transcripts (transcription start sites) and allows investigating promoter structures necessary for gene expression. Patients with FLT3-ITD/D835 harbored a higher number of co-mutations such as ASXL1 and RUNX1 compared to AML with FLT3-ITD (FLT3-ITD/D835: 2.83 ± 0.52, FLT3-ITD: 0.49 ± 0.13, p 〈 0.0001). Intriguingly, CAGE detected significantly higher expression of the anti-apoptotic Bcl-2 family genes BCL2 and BCL2A1 in FLT3-ITD/D835 compared to FLT3-ITD mutant primary samples. Specifically, the CAGE peak of BCL2 was highest in samples with FLT3-ITD/D835 alone (p 〈 0.01), while the CAGE peak of BCLA1 was highest in samples with FLT3-ITD/D835 and co-mutations compared with the other samples (p=0.01). To recapitulate the observations obtained with primary human AML samples, we generated MV4;11 cells with acquired FLT3-ITD/D835 mutations (MV4;11-QR cells) by culturing FLT3-ITD MV4;11 leukemia cells in the presence of quizartinib (1.5 nM), a selective FLT3 inhibitor, for 6 months. While quizartinib (0.2 nM) suppressed the proliferation of 50% of the parental MV4;11 at 72 hours, much higher concentrations of quizartinib (10 nM) was required to suppress the proliferation of MV4;11-QR cells. Quantitative RT-PCR and immunoblot analysis revealed that MV4;11-QR cells expressed higher transcript and protein levels of BCL2A1 than MV4;11 parental cells, while BCL2 levels were similar in both cells and MCL1 and BCLxL expression were lower in the MV4;11-QR than in the parental cells. Next, to investigate the molecular properties of AML cells bearing FLT3-ITD or FLT3-ITD/D835 without other co-mutations, we created Ba/F3 cells stably expressing FLT3-ITD or FLT3-ITD/D835. Of notes, the FLT3-ITD/D835 Ba/F3 cells expressed markedly higher BCL2 transcript and protein levels with lower expression of BCLxL than in FLT3-ITD Ba/F3 cells. No significant difference of MCL1 expression was observed. The sensitivity to quizartinib was massively decreased in the FLT3-ITD/D835 Ba/F3 cells (IC50: FLT3-ITD/D835 〉 1000nM vs. FLT3-ITD, 0.8nM, at 48h). Finally, we examined the efficacy of the BCL-2 specific inhibitor venetoclax in FLT3-ITD/D835 dual mutated cells with or without upregulation of BCL2 or BCL2A1, the latter shown to confer resistance to venetoclax by sequestering released BIM (Esteve-Arenys, Oncogene. 2018). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in BCL2 upregulated FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells (IC50: FLT3-ITD/D835 301nM vs. FLT3-ITD 〉 1000 nM, 96 h). However, FLT3-ITD/D835 bearing MV4;11-QR cells with upregulated BCL2A1 were less sensitive to venetoclax than MV4;11 parental cells (IC50: MV4;11-QR, 149nM vs. MV4;11, 33 nM, 72 h). In conclusion, these results demonstrate that acquisition of D835 mutation in FLT3-ITD mutated AML is often accompanied with multiple co-occurring genetic mutations, and depends on anti-apoptotic BCL-2 associated pro-survival mechanisms. BCL2A1 upregulation might be involved in pathogenesis of acquired drug resistance of FLT3-ITD/D835 dual mutant AML cells, and is a promising new target in FLT3-ITD/D835 refractory AML with complex mutations. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Shah:Bristol-Myers Squibb: Research Funding. Konopleva:Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.