The implementation of targeted therapies for acute myeloid leukemia has been challenged by complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here, we report initial findings from the Beat AML program on a cohort of 672 tumor specimens collected from 562 patients. We assessed these specimens using whole exome sequencing, RNA-sequencing, and ex vivo drug sensitivity analyses. Our data reveal Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Potent FLT3 inhibitors such as quizartinib (AC220) have shown promise in treating acute myeloid leukemia (AML) containing FLT3 internal tandem duplication (ITD) mutations. However, responses are not durable and resistance develops within months. In this study, we outline a two-step model of resistance whereby extrinsic microenvironmental proteins FLT3 ligand (FL) and fibroblast growth factor 2 (FGF2) protect FLT3-ITD+ MOLM14 cells from AC220, providing time for subsequent accumulation of ligand-independent resistance mechanisms. FL directly attenuated AC220 inhibition of FLT3, consistent with previous reports. Conversely, FGF2 promoted resistance through activation of FGFR1 and downstream MAPK effectors; these resistant cells responded synergistically to combinatorial inhibition of FGFR1 and FLT3. Removing FL or FGF2 from ligand-dependent resistant cultures transiently restored sensitivity to AC220, but accelerated acquisition of secondary resistance via reactivation of FLT3 and RAS/MAPK signaling. FLT3-ITD AML patients treated with AC220 developed increased FGF2 expression in marrow stromal cells, which peaked prior to overt clinical relapse and detection of resistance mutations. Overall, these results support a strategy of early combination therapy to target early survival signals from the bone marrow microenvironment, in particular FGF2, to improve the depth of response in FLT3-ITD AML.

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Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.

Key Points• FGF 2 promotes IM resistance in vitro and in vivo and is overcome by ponatinib, an FGF receptor and ABL kinase inhibitor.Development of resistance to kinase inhibitors remains a clinical challenge. Kinase domain mutations are a common mechanism of resistance in chronic myeloid leukemia (CML), yet the mechanism of resistance in the absence of mutations remains unclear. We tested proteins from the bone marrow microenvironment and found that FGF2 promotes resistance to imatinib in vitro. Fibroblast growth factor 2 (FGF2) was uniquely capable of promoting growth in both short-and long-term assays through the FGF receptor 3/ RAS/c-RAF/mitogen-activated protein kinase pathway. Resistance could be overcome with ponatinib, a multikinase inhibitor that targets BCR-ABL and FGF receptor. Clinically, we identified CML patients without kinase domain mutations who were resistant to multiple ABL kinase inhibitors and responded to ponatinib treatment. In comparison to CML patients with kinase domain mutations, these patients had increased FGF2 in their bone marrow when analyzed by immunohistochemistry. Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGF receptor inhibition. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance. (Blood. 2014;123(10):1516-1524