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Introduction: CPX-351 (Vyxeos), a liposomal combination of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio, has recently been shown to be significantly more efficacious than the standard of care, 7+3 administration regimen of free (non-liposomal) cytarabine + daunorubicin in treating elderly high-risk AML patients, including patients with the FLT3-ITD mutation. We previously exposed AML blasts from primary patient samples to CPX-351 ex vivo and found that samples harboring FLT3-ITD were significantly more sensitive to CPX-351 and also demonstrated enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in upregulated liposome uptake pathways, leading to increased cell death and overall sensitivity to CPX-351. Furthermore, we wanted to examine the effect of combining CPX with existing FLT3 inhibitors (e.g. quizartinib or midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains mutant, activated FLT3) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We measured cell viability and intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry and using a fluorescent plate reader. Additionally, we performed synergy analyses on the cytotoxic activity in these cells, adding CPX-351 and FLT3 inhibitors in combination at different exposure sequences followed by measurement of cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351, and exhibited increased drug uptake, compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, indicating that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, in synergy studies we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These results provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351, which is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Disclosures Tardi: Jazz Pharmaceuticals: Employment. Mayer:Jazz Pharmaceuticals: Employment.