An important limitation of FLT3 tyrosine kinase inhibitors (TKIs) in the treatment of FLT3-ITD positive AML is the development of resistance. Two FLT3-TKIs were recently approved by the FDA. Midostaurin improved overall survival when added to standard chemotherapy in newly diagnosed FLT3 mutated AML.1 The second generation FLT3 kinase inhibitor gilteritinib showed improved overall survival in relapsed/refractory FLT3-ITD positive AML compared to standard salvage therapy.2 A widespread use of those inhibitors emphasizes the need to better understand resistance towards FLT3 directed therapies. Secondary FLT3 (TKD) mutations are associated with clinical resistance towards FLT3 inhibitors.3 In vitro, we noted that the majority of FLT3 inhibitor-resistant FLT3-ITD cell lines did not harbor secondary FLT3 resistance mutations.3 In this study, we explored FLT3-ITD kinase-independent resistance and discovered a JAK1 V658F mutation in about 30% of cell lines with acquired resistance to midostaurin or sorafenib. We discovered CSF2RB, the common receptor beta chain of IL-3, IL-5 and GM-CSF as a signaling intermediate of FLT3-ITD that can be reactivated by the newly acquired JAK mutation and thus mediates FLT3-ITD independent STAT5 phosphorylation.
To investigate whether JAK family mutations contribute to treatment resistance in AML patients treated with FLT3 inhibitors, we sequenced FLT3 and JAK1-3 in samples from 136 FLT3 mutated AML patients who had undergone FLT3 inhibitor treatment. In 136 patients with FLT3-ITD positive AML and resistance, we found mutations in JAK1-3 in six patients (4.4%) and secondary FLT3 mutations in 13 patients (9.6%). Importantly, secondary FLT3 mutations and JAK1-3 mutations, except for one patient, occurred in a mutually exclusive manner. Therefore, JAK mutations might represent an alternative, FLT3 independent mechanism of clinical resistance to FLT3-TKIs in AML.
In vitro, we were able to demonstrate that pharmacological inhibition of both FLT3 and JAK kinases abolished cell growth and downstream signaling in FLT3-ITD positive cells expressing one of the activating JAK mutations identified in our patient cohort. The combination of the FLT3 inhibitors midostaurin or gilteritinib together with the JAK1/2 inhibitor ruxolitinib had strongest activity to overcome resistance promoted by activating JAK muations. In concert with other recent evidence demonstrating that inhibition of JAK kinases can help override stroma-based resistance in FLT3 mutated AML,4,5,6 we deem combination treatment with a FLT3- and JAK-inhibitor an attractive strategy to overcome resistance towards FLT3 TKIs mediated by FLT3-independent JAK-STAT activation, especially in cases where activating JAK family mutations are found.
In summary, these data indicate that in FLT3-mutated AML, rarely emerging, activating JAK family mutations contribute to FLT3 independent resistance to therapeutic FLT3 inhibitors. Therefore, patients with FLT3 mutated AML relapsing after exposure to FLT3 inhibitors should be tested for JAK family mutations and might benefit from dual FLT3 and JAK inhibitor treatment.
- Stone RM, Mandrekar SJ, Sanford BL, Laumann K, Geyer S, Bloomfield CD, et al. Midostaurin plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med. 2017 03;377(5):454–64.
- Perl AE, Martinelli G, Cortes JE, Neubauer A, Berman E, Paolini S, et al. Gilteritinib or Chemotherapy for Relapsed or Refractory FLT3-Mutated AML. N Engl J Med. 2019 Oct 31;381(18):1728–40.
- Smith CC, Paguirigan A, Jeschke GR, Lin KC, Massi E, Tarver T, et al. Heterogeneous resistance to quizartinib in acute myeloid leukemia revealed by single-cell analysis. Blood. 2017;130:48–58.
- Sung PJ, Sugita M, Koblish H, Perl AE, Carroll M. Hematopoietic cytokines mediate resistance to targeted therapy in FLT3-ITD acute myeloid leukemia. Blood Adv. 2019 Apr 9;3(7):1061–72.
- Patel AB, Pomicter AD, Yan D, Eiring AM, Antelope O, Schumacher JA, et al. Dasatinib overcomes stroma-based resistance to the FLT3 inhibitor quizartinib using multiple mechanisms. Leukemia. 2020 May 14;1–11.
- Weisberg E, Liu Q, Nelson E, Kung AL, Christie AL, Bronson R, et al. Using combination therapy to override stromal-mediated chemoresistance in mutant FLT3-positive AML: synergism between FLT3 inhibitors, dasatinib/multi-targeted inhibitors and JAK inhibitors. Leukemia. 2012 Oct;26(10):2233–44.