Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell–derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

While clinical benefit has been observed with gilteritinib in patients with FLT3 mutated relapsed/refractory acute myeloid leukemia (AML), most patients relapse through mechanisms that are incompletely understood. In this study, to investigate mechanisms of gilteritinib sensitivity and resistance, we performed targeted sequencing (21 patients) and scRNASeq analysis (8 patients) of FLT3-ITD-positive AML samples obtained before and during treatment. Before treatment, co-occurring mutations were observed in 33 genes among 21 patients. Mutations in RAS pathway genes (PTPN11, KRAS, NRAS, CBL) were the most common and observed in 57% (12/21) of patients. Seven patients pretreatment already contained RAS pathway mutations, of which 6 of these mutations were maintained over the course of treatment. During treatment, 9 patients showed emerging RAS mutations, 4 of which initially presented with a different RAS pathway mutation pre-treatment. Other mutations that arose during treatment were observed in CEBPA, IDH1, SF1 and WT1; as well as CSF3R, CUX1, PLEKHG5, and XPO1, not previously identified in gilteritinib-treated patients. Mutational clonality was generally maintained over treatment in both responders and non-responders. scRNASeq revealed global gene expression differences in myeloblast populations between gilteritinib-responsive and -unresponsive patients. Previous studies in vitro have shown that bone marrow-derived hematopoietic and inflammatory cytokines/chemokines confer resistance to FLT3 inhibitors. In the unresponsive group, we observed an increase in expression of CCL5, CXCL1, CXCL2, CXCL8, FLT3, IL6R, IL3RA, and CSF2RA during gilteritinib treatment, supporting the concept from preclinical studies that AML microenvironment-mediated factors play a critical role in drug resistance. Baseline expression of the Tec kinase BMX was significantly higher in unresponsive patients (Log2FoldChange, 6.65; adjusted P value, 0.00186), and this was maintained in the expanding myeloblast populations during treatment. Previously, upregulated BMX was shown to contribute to sorafenib resistance in patients with FLT3-ITD-positive AML, through cell-nonautonomous microenvironment hypoxia-dependent effects. Further in vitro investigation confirmed gilteritinib resistance could be reversed through genetic and pharmacological manipulation of BMX. Gene module analysis showed associations between gilteritinib responsive and upregulation of genes and pathways involved in lymphocyte differentiation and myeloid leukocyte activation, including TBX21, GATA3, CD33, and LYZ. By contrast, there was association between unresponsiveness to gilteritinib and upregulation of cell-cycle, DNA, and RNA metabolic processes, including pathways involving METTL1 and DNMT3A, as well as pre-treatment expression of pathways associated with protein translation. Together, these data provide support for microenvironment-dependent escape from targeted therapy and suggest that BMX may contribute to gilteritinib resistance. High-dimensional analysis with scRNA-seq provides a deeper understanding of targets and pathways for potential therapeutic intervention to restore gilteritinib sensitivity. Disclosures Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria.

BackgroundThe development of tyrosine kinase inhibitors (TKIs) for the treatment of acute myeloid leukemia (AML) with FLT3 internal tandem duplication mutations (FLT3‐ITD+) has been challenging due to intrinsic and acquired drug resistance. Important resistance mechanisms that have been described include: 1) the emergence of secondary FLT3 tyrosine kinase domain (TKD) mutations; 2) compensatory signaling pathways (intrinsic to the leukemia cell or due to tumor‐microenvironment interactions), such as Gas6/Axl upregulation; and 3) the co‐occurrence of somatic mutations with FLT3‐ITD, such as IDH1/2, NRAS and MLL‐PTD. TP‐0903, is a new TKI in development as an Axl inhibitor. Previously, we demonstrated that TP‐0903 is a potent inhibitor of FLT3‐ITD and drug resistant FLT3 TKD mutations in preclinical in vitro and in vivo models (Jeon JY et al. ASH 2017). Here, we present the activity of TP‐0903 in additional TKI‐resistant models of FLT3‐ITD+ AML in comparison to other FLT3 TKIs.MethodsFLT3‐ITD+ MOLM13 cells were seeded in direct co‐culture with or without HS5‐GFP bone marrow stromal cells (MSCs) for 24h followed by 72h treatment with TP‐0903 or 5 other clinical candidate FLT3 TKIs; cell viability of co‐cultured MOLM13 cells was measured via MTT assay. Soluble Gas6 and Axl were measured in co‐cultured media over 24–72h by ELISA. Ex vivo activity of TKIs on the viability of murine primary leukemia cells with FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD co‐occurring mutations was assessed after 72h TKI treatment using a CellTiter Glo assay.ResultsThe effect of TP‐0903 and other FLT3 TKIs on the inhibition of viability of MOLM13 cells cultured with or without MSCs is shown in Table 1. While co‐culture induced a minimal 1.7‐fold shift in the TP‐0903 IC50 value, the other FLT3 TKIs were more resistance in co‐culture, with gilteritinib and sorafenib showing the greatest change in IC50 values (4.9‐ and 5.0‐fold). Soluble Axl and Gas6 levels increased over 24–72h in co‐cultured media with the highest concentrations achieved of 1455 ± 322 pg/mL and 5098 ± 974 pg/mL, respectively. In primary FLT3‐ITD+/−/IDH2‐R140Q+/− leukemia cells, TP‐0903 was 8‐ to 10‐fold more potent than gilteritinib and crenolanib, whereas midostaurin, quizartinib and sorafenib did not show activity in these cells (Table 1). TP‐0903 was also 5‐fold more potent than gilteritinib in primary FLT3‐ITD+/−/MLL‐PTD leukemia cells. Ongoing studies are evaluating the effect of soluble Gas6 on TKI sensitivity in AML cells, as well as the in vivo activity of TP‐0903 in FLT3‐ITD+/−/IDH2‐R140Q+/− or FLT3‐ITD+/−/MLL‐PTD spontaneous mouse models.ConclusionsTP‐0903 retains activity in preclinical models of intrinsic/acquired FLT3 TKI resistance with therapeutic potential to overcome drug resistant FLT3‐ITD+ AML.Support or Funding InformationThis work is supported by Eli Lilly fellowship and NIH grant 5R01CA138744‐09This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Clonal heterogeneity and co‐occurring mutations contribute to drug resistance in AML. Leukemia samples from patients progressing on gilteritinib, a FLT3 inhibitor, were selected for RAS mutant clones, which are known to contribute preclinically to resistance (McMahon CM, Cancer Discov 2019). Previously, we demonstrated that TP‐0930, a multi‐kinase inhibitor, targets FLT3‐ITD and resistance‐conferring tyrosine kinase domain mutations in preclinical models. Here, we present the activity of TP‐0903 in RAS pathway mutant models of AML. KiNativ assay was performed in OCI‐AML3 cells (NRAS Q61L) treated with TP‐0903 (100 nM, 2h). Inhibition of cell viability was evaluated in AML cell lines (MTT) and primary samples (CellTiter Glo). Inhibition of AURKA/B, AKT, ERK, MCL‐1, and BCL‐2 were determined by western blot. Apoptosis, cell cycle, and differentiation assays were performed in OCI‐AML3 using annexin V, DAPI and CD11b‐APC staining. In vivo activity of TP‐0903 was evaluated in a systemic OCI‐AML3‐Luc+ xenograft model; 5e5 luciferase expressing cells were tail vein injected to NSG mice and TP‐0903 50 mg/kg or vehicle was given orally once dailyx5/week. Progression of leukemia was monitored via bioluminescence imaging and Kaplan Meier was used for survival analysis. In Kinativ assay, inhibition of native kinases were similar to those observed in a previous binding assay. We observed that TP‐0903 inhibited ACK1 (activated CDC42 kinase 1 or TNK2) and GCK (germinal center kinase or MAP4K2) by 87 and 46%, respectively. Inhibition of ACK1 (Kd = 7.3 nM, IC50 = 30.7 nM) and GCK (Kd = 1.8 nM, IC50 = 2.5 nM) was confirmed in binding and kinase assays. In RAS mutant AML cell lines (OCI‐AML3, THP1, HL60, and U937), TP‐0903 inhibited viability with IC50 values of 26 – 99 nM. In OCI‐AML3, TP‐0903 at 100 nM inhibited pAURKA/B, pAKT and MCL‐1; caused a G2‐M arrest and polyploidy (20 nM, 24h); and induced apoptosis (20–50 nM, 72h). TP‐0903, compared to gilteritinib, was more potent in inhibiting 4 ex vivo human primary AML samples with N/KRAS mutations co‐occurring with wt FLT3 or FLT3‐ITD, along with other mutations (IC50 range, 21 – 60 vs 260 – 1370 nM). In a 14‐day CFU assay, TP‐0903 inhibited colony formation of a FLT3‐ITD and NRAS mutant primary sample compared to DMSO (mean, 65 vs 188 CFUs). In an OCI‐AML3‐Luc+ xenograft study, TP‐0903 suppressed the outgrowth of leukemia at day 21 (P<0.0001) and prolonged median survival by 9 days compared to veh‐treated mice (P<0.0001). At study endpoint, spleen weight was higher in veh‐treated mice than in TP‐0903‐treated mice (mean ± SD, 333 ± 23 vs 181 ± 21 mg, P=0.0029). TP‐0903, a novel multi‐kinase inhibitor, shows promising in vitro and in vivo activity in RAS mutant AML. In a previous report (Nonami A, Blood 2015), an integrated approach involving cell‐based pharmacologic screening combined with KiNativ, gene expression profiling and mechanism studies, identified 2 kinase signaling pathways (ACK1/AKT and GCK) that synergistically contributed to the growth of NRAS mutant leukemia cells, i...

The tyrosine kinase inhibitor (TKI) gilteritinib has recently been granted FDA approval for the treatment of relapse/refractory FLT3‐ITD+ AML, yet its mechanism of action is not entirely known. Two recent studies investigating metabolic adaptations during treatment with the FLT3 TKI quizartinib in AML have demonstrated reduced glycolysis or impaired glutaminolysis. Although these studies are conflicting, it suggests that TKIs may influence metabolic changes thereby impacting proliferation and cell viability. Many highly proliferative cells require glutamine as a precursor since they depend on the glutaminolysis pathway for generating building blocks and energy for anabolic processes. Here, we hypothesize that gilteritinib reduces cell viability in FLT3‐ITD+ AML by altering glutamine uptake and metabolism. In our preliminary studies, mice with FLT3‐ITD+ AML (MOLM13 xenograft) were treated with vehicle or gilteritinib (30 mg/kg once daily, five days/week) until leukemic progression. Using an unbiased transcriptomic approach (RNA‐seq) of the leukemic bone marrow, we found that the glutamine transporters (SLC38A1, SLC1A5 and SLC7A5) were significantly downregulated in the gilteritinib‐treated cohort compared to the vehicle cohort (fold change 1.96–4.70, P < 0.0001). Furthermore, expression of metabolic genes was altered in glutamine/aspartate metabolism, glycolysis, and the TCA cycle. Using a resistant xenograft model in which the FLT3‐ITD harbors a mutation in the tyrosine kinase domain at residue D835Y (MOLM13‐RES), SLC38A1 and SLC7A5 were also significantly downregulated after gilteritinib treatment (fold change 0.70–1.28, P < 0.0005), as well as having alterations in the gene expression profile of genes in the aforementioned pathways, although to a lesser extent than MOLM13. In vitro, depleting the cell culture medium of glutamine decreases the cell viability by ~50% in MOLM13 and another FLT3‐ITD+ AML cell line (MV4‐11), indicating that these leukemic cells are addicted to glutamine metabolism as an energy and precursor source. Acute exposure of MOLM13 cells to 100 nM FLT3 TKIs gilteritinib, quizartinib and sorafenib all decrease the transcript of glutamine transporters, with the greatest response from gilteritinib as measured via RT‐PCR (P < 0.01 for all three transporters). In cellular uptake assays, the uptake of glutamine by MOLM13 cells was decreased stepwise to nearly 50% during an 8 h timecourse of 100 nM gilteritinib treatment. This was confirmed using unbiased isotope labeling and tracing in vitro in MOLM13 and MOLM13‐RES cells, which showed reduced glutamine uptake and utilization through the TCA cycle after gilteritinib treatment (15 nM, 24 h). Metabolomics of MOLM13 in vivo samples also revealed alterations in the metabolic pathways. Thus, our data support the premise that gilteritinib downregulates glutamine transporters and alters glutamine metabolism to inhibit cell viability of FLT3‐ITD+ AML. Support or Funding Information National Cancer Institute under award R01 CA138744‐08 and the Ohio State U...