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L. Lee et al.
cytochrome P450 enzymes within stromal cells and the production of cytokines that activate pro-survival path- ways within the leukemia cells.10,28 Overcoming the pro- tective effect of marrow could lower disease burden in responding patients, and either directly prolong survival or allow more successful application of allogeneic transplan- tation.
We have turned to BET inhibitors to undermine the stroma-derived maintenance of pro-survival proteins in blasts within the marrow. BET inhibitors have attracted considerable interest as potential therapies for different diseases, but it seems unlikely that sustained suppression of BET protein activity would be tolerated in humans, a concern that has largely been borne out by early studies of first generation BET inhibitors.29,30 However, PLX51107 has a relatively short plasma half-life in human patients, and our ex vivo surrogate PIA assay for MYC indicates effective BET inhibition can be achieved with this drug even at modest daily doses. Using the MYC PIA assay, in combination with the FLT3 PIA assay, a plasma sample from a patient taking both a BET and FLT3 inhibitor can be assayed for each drug effect separately, using OCI- AML3 cells for BET inhibition and MOLM-14 cells for the FLT3 inhibition. Even though intermittent, the BET inhibi- tion induced by PLX51107 still results in synergistic cyto- toxicity in combination with FLT3 inhibition against blasts on BM stromal cells. BET inhibition is expected to affect MYC and a myriad of other key genes, any number of which could contribute to the synergistic effect.18 Our use of MYC in the PIA assay is primarily a surrogate for BET activity, and, indeed, our data indicate that it is likely to be the most appropriate.
In designing a clinical protocol for this combination reg- imen, we will need to choose a clinically validated FLT3
inhibitor. Both gilteritinib and quizartinib prolong survival for relapsed FLT3-ITD AML, and both are synergistic with BET inhibition. FLT3-TKD mutations emerge in response to monotherapy with quizartinib,11 while RAS mutations emerge in response to gilteritinib therapy,31 so neither drug offers a particular advantage in this regard. Quizartinib is more myelosuppressive than gilteritinib,27 but, at least in our colony assays, the addition of BET inhibition does not seem to increase this. On the other hand, quizartinib, as a type II inhibitor, is more selective than gilteritinib, and its combination with another small molecule inhibitor may result in fewer off-target effects.
In summary, this work provides the scientific founda- tion for a clinical trial of a BET plus FLT3 inhibitor for the treatment of relapsed/refractory FLT3-ITD AML. The PIA assays for MYC and FLT3 can provide laboratory corre- lates to quantify in vivo inhibition of both targets in the dose escalation and expansion cohorts.
Disclosures
ML is a consultant to Daiichi-Sankyo, Astellas, Novartis, FujiFilm, Amgen, and Agios, and receives research funding from Astellas, FujiFilm, and Novartis; PS, BP, CZ, YM and GB are employees of Plexxikon, whose product, PLX51107, is a subject of this research; YH, MN and HS are employees of Daiichi- Sankyo, whose product, quizartinib, is a subject of this research; LL, DH and TR have no conflict of interest to disclose.
Contributions
LL performed experiments, analyzed data and wrote the manuscript; YH, PS, BP, CZ, YM, MN, HS, DH, TR and GR performed experiments, analyzed data and edited the manu- script; ML designed the study, analyzed data and wrote the manuscript.
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