Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children’s Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor.

In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance.

Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells.

Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients.