PTPN11 Mutated Acute Myeloid Leukemia (AML) Features an Abundant Epichaperome Network and Is Sensitive to the Epichaperome Inhibitor PU-H71

PTPN11 mutations occur in roughly 5-10% of AML patients. There is no defined impact on prognosis in the literature, although a single center analysis from our group suggested an association with poor outcomes. The pathogenesis of activating PTPN11 mutations is hypothesized to result in increased signaling through RAS resulting in uncontrolled cellular proliferation.

PU-H71 is an epichaperome inhibitor that has shown pre-clinical promise in ex-vivo and PDX models of AML (Cell Reports 2015). The efficacy of the drug is dependent on the tumor possessing epichaperomes, hyperconnected networks of chaperones and co-chaperones facilitating tumor survival (Nature 2016, Nature Medicine 2018). Prior studies have found an association between epichaperome abundance and overactivity of the transcription factor MYC. Activating PTPN11 mutations have also been shown to result in MYC overactivity through increased SHP2 signaling (protein product of PTPN11 gene) and thus we hypothesized that through increased activity of MYC, activating PTPN11 mutations in AML lead to an abundant epichaperome network, and in turn render these tumors sensitive to the epichaperome inhibitor, PU-H71.

15 PTPN11 mutated AML peripheral blood and bone marrow samples from two academic medical centers (Weill-Cornell Medical Center and University of Colorado) were collected to be analyzed for epichaperome abundance and sensitivity to the epichaperome inhibitor PU-H71. To detect epichaperome abundance, PTPN11 mutated AML samples were incubated with FITC-bound PU-H71 (labeled F2) as well as a chemically altered FITC- bound PU-H71 "control" (labeled F9) which does not bind the epichaperome (Bioorg Med Chem Lett 2011). The AML cell line MV411 was used as a positive control (given known epichaperome abundance and sensitivity to PU-H71) and intra-sample T-cells were used as negative controls. These samples were analyzed using multi-parameter flow cytometry including markers to distinguish blasts, progenitors and lymphocytes. For analysis of sensitivity to PU-H71, samples were incubated with two different concentrations of PU-H71, 1uM and 0.5uM, for 48 hours and subsequently evaluated for viability using multi-parameter flow cytometry (including markers to distinguish blasts, progenitors and lymphocytes). Leukemic blasts and T cells were gated based on immunophenotype, and the ratio of blast F2 to F9 was combined with the F2-F9 blast to T-cell ratio to calculate epichaperome abundance, using a cut-off value of 4.5 to determine medium to high or low levels of the epichaperome (medium to high, >/=4.5, low, <4.5).

Of the 15 samples analyzed for epichaperome abundance. Seven out of 15 (46.7%) samples showed an abundant epichaperome. Nine of these samples had complete viability data, of which, five out of nine (55.5%) showed >60% cell death after 48 hour incubation with PU-H71. For these nine samples with data on sensitivity to PU-H71, the presence or absence of epichaperome abundance was predictive of ex vivo sensitivity (5 sensitive, 4 resistant).

We have established PDX-mouse models to evaluate in vivo sensitivity to PU-H71, using epichaperome abundant samples. We have confirmed the epichaperome levels in AML blast cells from the peripheral blood of the animals. Currently the treatment and analysis is ongoing.

These data suggest that PTPN11 mutated AML represents a subset of AML that could be particularly sensitive to epichaperome inhibition. Additionally, the findings from our studies suggest this assay has promise as a means to accurately predict sensitivity to PU-H71, a useful precision medicine tool for a difficult to treat disease. All samples are currently being analyzed for MYC transcriptional activity and other signaling pathways.