Insights in Dynamic Kinome Reprogramming As a Consequence of MEK Inhibition in MLL-Rearranged AML.

Abstract 2338

Cancer arises when somatic cells are able to escape the restraints that normally withhold them from unlimited expansion. Cancer progression is thought to be the net result of signaling through various protein-kinase mediated networks driving cell proliferation and survival. The kinome networks can be affected by numerous factors; including acquired or selected mutations as well as environmental cross talk. Additionally, the loss of phosphatases could be a causative factor for activation of multiple tyrosine kinases as well (Cell. 2011 Mar 4;144(5):703–18).

Deregulated kinase activity is frequently observed in leukemia, leading to induced proliferation, migration, survival, and chemotherapy resistance of leukemic cells. (Leuk Lymphoma. 2011 Jan;52(1):122–30, Leukemia. 2005 Apr;19(4):586–94, Exp Hematol. 2005 Jun;33(6):660–70) However, single kinase-targeted cancer therapies can default when cancer cells bypass through alternative routes, facilitating therapeutic resistance. In order to circumvent the constraints given by an inihibitor, we need to monitor kinome reprogramming upon mono-treatments to develop the most successful combination therapy approach for disease specific subgroups, as poor prognostic MLL-rearranged AML. Rational designs of kinase inhibitor or RTK antibody combinations require a high-throughput measurement of kinome and proteome activity signatures within this patient subgroup. Figure 1 outlines our approach to explore the intracellular signaling networks and study the dynamic changes resulting in reprogramming of the kinome network, with the goal to define combinational therapeutic strategies.

In this study, we succeeded using combined high-throughput approaches for kinomic and proteomic profiling to identify specific aberrant kinase signatures in MLL-rearranged AML as compared to NBM (Fig. 1B/C). The altered activated kinase signatures of a comprehensive set of MLL-rearranged AML patient samples resulted in a detailed map of the overall kinase activity and phosphorylation of signal transduction molecules, which allowed the selection of possible druggable targets i.e. MEK, JNK, and CREB (Fig. 1B/C). Pharmacological MEK inhibition in primary MLL-rearranged AML demonstrated to be most successful in reducing the AML cell survival, without showing cytotoxicity in NBM (mean MEK inhibitor IC50 of 3.5μM ⁺/_- 0.7μM in primary MLL-rearranged AML versus a mean IC50 >50μM in NBM), whereas for CREB and JNK inhibitors MLL-rearranged AML cells were equally affected as NBM cells. Dynamic kinome reprogramming of signaling networks in response to MEK therapy did occur, by inducing the activation of RTKs to bypass the initial MEK inhibitory effects in a MLL-rearranged AML cell line. The dynamic escape mechanism allowed us to predict and test the efficacy of novel combination strategies. Combined MEK and VEGFR-2 inhibition demonstrated to induce cell death sufficiently in MLL-rearranged AML (Fig. 1D). This advantageous strategy allows rational design of successful and selective combination therapies for specific target inhibitors.

Figure 1.

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Kinomics, proteomics, and signaling dynamics for novel combination therapies in MLL-rearranged AML

(A) Overall study design for novel combination therapies (B) Constructed kinome trees showing the overall kinase derived peptide activity in 15 individual primary MLL-rearranged AML samples versus 5 NBM samples to identify the distribution and the overlap of kinase activity. Combined kinome trees revealed induced JNK annotated peptide activity in MLL-rearranged AML. (C) Representative protein kinase phosphorylation arrays of primary MLL-rearranged AML (n=9 in total) versus NBM (n=4 in total). Phosphoproteomic analysis identified induced phosphorylation levels of MEK and CREB protein kinases in MLL-rearranged AML. (D) Evaluation of AML cell survival of an anticipated successful combined therapeutic strategy in MLL-rearranged AML, showing the synergistic beneficial effect of combined MEK and VEGFR-2 inhibition in MLL-rearranged AML cell line THP-1.

Figure 1.

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Kinomics, proteomics, and signaling dynamics for novel combination therapies in MLL-rearranged AML

(A) Overall study design for novel combination therapies (B) Constructed kinome trees showing the overall kinase derived peptide activity in 15 individual primary MLL-rearranged AML samples versus 5 NBM samples to identify the distribution and the overlap of kinase activity. Combined kinome trees revealed induced JNK annotated peptide activity in MLL-rearranged AML. (C) Representative protein kinase phosphorylation arrays of primary MLL-rearranged AML (n=9 in total) versus NBM (n=4 in total). Phosphoproteomic analysis identified induced phosphorylation levels of MEK and CREB protein kinases in MLL-rearranged AML. (D) Evaluation of AML cell survival of an anticipated successful combined therapeutic strategy in MLL-rearranged AML, showing the synergistic beneficial effect of combined MEK and VEGFR-2 inhibition in MLL-rearranged AML cell line THP-1.

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