Combining Dasatinib and AC220 Reduces Stroma-Based pSTAT5Y694 in FLT3-ITD+ AML and Overcomes FLT3 TKI Resistance
Authors: Patel Ami B. , Pomicter Anthony D. , Yan Dongqing , Eiring Anna M. , Franzini Anca , O'Hare Thomas , Deininger Michael W. 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with a poor prognosis. FLT3 internal tandem duplications (ITDs) are found in ~30% of AML patients and are associated with inferior survival. Addition of FLT3 tyrosine kinase inhibitors (TKIs) such as midostaurin improve survival of AML treated with cytotoxic chemotherapy. However, relapse rates remain unacceptably high. Protection by stroma-derived survival signals is thought to enable survival of leukemia initiating cells (LICs) in the presence of FLT3 TKIs, providing a reservoir for subsequent overt relapse. We previously reported that HS-5 conditioned medium (CM) rescued FLT3-ITD positive AML cell lines and primary cells from the effects of the 2nd generation FLT3 TKI AC220 (quizartinib) as compared to cells cultured in regular medium (RM). Earlier work from our lab and others has demonstrated that HS-5 CM increases pSTAT3Y705 in chronic myeloid leukemia cells treated with BCR-ABL1 TKIs, leading to TKI resistance, while pSTAT5Y694 levels remain under the control of the BCR-ABL1 kinase (Eiring et al, Leukemia, 2015). Similar to BCR-ABL1, FLT3 signaling results in potent pSTAT5Y694 activation. We hypothesized that STAT3 and/or STAT5 activation contributes to stroma-mediated protection of FLT3-ITD+ AML cells upon FLT3 inhibition with AC220.
We found that FLT3-ITD+ cell lines and primary cells grown in HS-5 CM exhibited strong upregulation of pSTAT3Y705 and pSTAT5Y694 that was unaffected by treatment with 10nM AC220. In RM, pSTAT3Y705 was undetectable irrespective of AC220 dose and pSTAT5Y694 was abolished by 10nM AC220. shRNA knockdown experiments of STAT3 and STAT5 in FLT3-mutated cell lines grown in CM and treated with AC220 showed that STAT5 knockdown alone consistently resulted in greater inhibition of cell proliferation than STAT3 knockdown alone. This suggests that in FLT3-ITD+ AML cells cultured in CM, upregulation of pSTAT5Y694 by stroma-derived soluble factors is the main contributor to AC220 resistance.
We decided to use a candidate approach to identify the upstream kinases leading to pSTAT5Y694, focusing on inhibitors of pathways previously implicated in STAT5 activation in AML. We used phosphoflow to quantify pSTAT5Y694 in MOLM-13 cells grown for 24 hours in CM in the presence of 10nM AC220 combined with ruxolitinib, dasatinib, ibrutinib, PD173074 (FGFR1i) or PRT062607 (SYKi). Results from this screen indicated that the combination of dasatinib and AC220 most effectively decreased pSTAT5Y694 in MOLM-13 cells cultured in CM (minimum dasatinib dose =100nM). To further validate dasatinib as a candidate for combination drug treatment, we performed cell proliferation experiments in MOLM-13 cells cultured in CM for 72h with graded concentrations of AC220 and the candidate inhibitors at a minimum candidate drug dose of 100nM. Of the four inhibitors tested, the combination of dasatinib and AC220 most significantly decreased the IC50 of AC220 in MOLM-13 cells grown in CM (IC50 AC220 alone: 11.09nM; IC50 AC220 + 100nM dasatinib: 0.41nM; p<0.05, n=3).
It has recently been reported that FLT3-ITD AML cells harbor a highly glycolytic phenotype that can be partially suppressed by FLT3 TKIs (Huntly et al, Blood, 2018). Our preliminary Seahorse experiments show that the combination of dasatinib and AC220 more effectively inhibits glycolysis in MOLM-13 cells than AC220 alone, with differential modulation in CM versus RM. These results suggest that dasatinib inhibits a metabolic pathway critical to maintaining glycolysis in MOLM-13 cells challenged by AC220-based FLT3 inhibition. Moreover, these data indicate that extrinsic signals contribute to metabolic reprogramming of FLT3+ AML cells, enabling survival despite FLT3 inhibition. Experiments are underway to delineate how CM contributes to metabolic resistance and how dasatinib intercepts this survival signal. Our results support the concept of combining AC220 with dasatinib to reduce residual leukemia in FLT3+ AML and prolong survival. Further investigation is ongoing and will be reported at the time of abstract presentation.

Disclosures
Deininger: Blueprint: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees.