SHP2 Inhibition Overcomes Adaptive and Acquired Resistance to FLT3 TKI to Improve Efficacy Against FLT3/ITD AML

FMS-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase (RTK) that is frequently mutated in acute myeloid leukemia (AML), with the internal tandem mutation (FLT3/ITD) conferring a poor prognosis to AML patients. This makes FLT3/ITD AML a validated target for treatment with tyrosine kinase inhibitors (TKI), but their success has been hindered by mechanisms of resistance including rapid signal reprogramming known as adaptive resistance and acquired resistance gained through long-term treatment. Past work from our lab has shown that FLT3/ITD cells undergo adaptive resistance, through the rapid reactivation of ERK signaling within 24 hours of sustained FLT3 inhibition. To improve therapy of FLT3/ITD AML it is important to identify therapeutic targets that overcome this ERK reactivation.

Since adaptive ERK reactivation is often due to the relief of negative feedback loops that result in the activation of other RTK, we performed an RTK phospho-array on the FLT3/ITD cell line MV4;11. 48-hour treatment with the FLT3 TKI sorafenib resulted in increased activation of IGF-R, HER3 and AXL. While recent work from our lab found that combining AXL and FLT3 inhibition abrogated the ERK reactivation and exerted greater antileukemic effects, the activation of multiple RTKs suggests that targeting a signaling hub that inhibits multiple RTKs at once will likely prove more efficacious in overcoming this adaptive response. Recent literature has implicated SH2-containing protein tyrosine phosphatase 2 (SHP2) as a therapeutic target in adaptive ERK reactivation in other cancers. SHP2 acts directly downstream of RTKs and is involved in the activation of the ERK pathway. Therefore, we explored whether the addition of an SHP2 inhibitorto FLT3 TKI can overcome the adaptive response and increase treatment efficacy.

To explore this possibility, we first treated the FLT3/ITD cell lines Molm14 and MV4;11 as well as FLT3/ITD patient samples with FLT3 TKI alone or in combination with an SHP2 inhibitor for 24 hours. The addition of SHP099 to sorafenib decreased the expected ERK reactivation in a dose-dependent manner. Furthermore, combination treatment with synergized to decrease proliferation and increase apoptosis in the FLT3/ITD cell lines. Impressively, in a long-term assay, treatment with the combination for just 72 hours greatly reduced leukemic cell recovery.

To determine whether this combination would improve efficacy in vivo, we engrafted NSG mice with Molm14 cells and treated them for 4 weeks via oral gavage. The combination of SHP099 with gilteritinib group resulted in a statistically significantly lower leukemia burden compared to either treatment alone and the vehicle control. The combination group also had prolonged survival compared to the other groups.

In addition to rapid adaptive resistance, there are mechanisms of acquired resistance that render traditional FLT3 TKI treatments ineffective against FLT3/ITD AML cells. We next examined the effects of SHP2 inhibition on FLT3-TKI resistant cells models that were generated by growing and maintaining Molm14 and MV4;11 cells in the presence of FLT3 TKI. The combination of FLT3 TKI and SHP099 resensitized the cells to FLT3 TKI, resulting in decreased ERK phosphorylation, decreased proliferation and increased apoptosis of these FLT3 TKI resistant cells.

We also explored the effects of treating the FLT3/ITD mutant cell lines with the combination of SHP099 and several of the cytotoxic chemotherapy agents typically used in the treatment of AML including cytarabine and daunorubicin. While daunorubicin or cytarabine alone had little effect on the level of phosphorylated ERK, the addition of SHP099 decreased ERK activation and the combination synergized to exert greater to decrease proliferation and increase apoptosis.

These studies reveal that combining FLT3 TKI treatment with an allosteric SHP2 inhibitor decreases the adaptive ERK reactivation observed in FLT3/ITD cells. The finding that SHP099 synergized with both FLT3 TKI and chemotherapy agents in different FLT3-mutated AML models speaks to the versatile efficacy of SHP2 inhibition in multiple AML models. Taken together, the data suggests that SHP2 inhibition can help overcome both adaptive and acquired resistance in FLT3/ITD AML and is a candidate to try to improve patient outcomes.