Chemical Genomic Screening Reveals That PI3K/mTOR Inhibition Enhances Activity of the Anti-Leukemia Stem Cell Compound Parthenolide.

Abstract 388

Leukemia stem cells (LSCs) have been shown to initiate and maintain AML. Given that LSCs have also been shown to be chemoresistant relative to the bulk leukemia population, LSCs are thought to provide a surviving reservoir of cells that drive disease relapse. This concept is further supported by studies suggesting that poor prognosis is associated with leukemias presenting with a higher percentage of LSCs. Thus, identification of new therapeutic regimens that target LSCs appears to be of clinical significance. Our previous efforts to target LSCs have demonstrated the capability of parthenolide (PTL), and its water-soluble clinical derivative, dimethylamino-parthenolide (DMAPT), to impair the survival and leukemogenic activity of phenotypically- and functionally-defined LSCs, respectively. In order to improve the clinical utility of PTL–based compounds, we sought to determine rational drug combinations that would enhance the efficacy of these agents in vivo. To this end, we turned to the use of chemical genomic screening. The transcriptional signature arising when primary patient specimens are exposed to PTL for 6 h reveals a significant “protective response” (i.e. augmentation of detoxifying enzymes, antioxidant responses, and the unfolded protein response). Given this observation, we hypothesized that compounds capable of impairing the protective response would synergize with PTL and its derivatives and thereby enhance their anti-leukemia activity. To test this hypothesis, we interrogated the Connectivity Map database for instances of compounds that produced maximal inhibition of the protective response at the gene expression level. Overwhelmingly, this screen indicated compounds acting along the PI3 kinase pathway including wortmannin, LY-294002, and rapamycin. Indeed, exposure of primary AML cells to the combination of wortmannin and sub-lethal doses of PTL significantly decreased viability of AML cells (24% viable) relative to PTL (65% viable) or wortmannin (66% viable) alone. Moreover, the combination resulted in a significant decrease in colony formation (Wort=67% , PTL=87.5 and PTL+Wort = 6% CFU relative to untreated). The effect of these combinations is synergistic and not additive (Chou-Talay method). Importantly, these effects remained confined to AML cells and not their normal hematopoietic counterparts. Furthermore, these observations were corroborated with rapamycin and temsirolimus. To examine molecular mechanisms underlying the enhanced anti-leukemia activity, immunoblot analyses were performed and demonstrated that the PTL-induced protective response was abolished by the combination of PTL with wortmannin or rapamycin, consistent with the chemical genomic prediction. Finally, to assess in vivo activity of the drug combination we employed the clinical agents DMAPT and temsirolimus in a mouse xenotransplant model. Briefly, NOD/SCID mice were injected with primary human AML cells and at three weeks post transplant began a three-week course of daily treatment (vehicle control, DMAPT alone, temsirolimus alone, or DMAPT+temsirolimus). Animals were sacrificed after treatment (i.e. six weeks post transplant) and tumor burden was evaluated in the bone marrow. The mean percent of leukemic cells for the combination of temsirolimus and DMAPT treatment resulted in a four-fold decrease which was significant when compared to either treatment alone (P<0.001). Together, these data indicate that inhibition of the PI3 kinase and/or mTOR pathway can enhance the anti-leukemia activity of parthenolide-based drugs. The studies also serve to emphasize the value of chemical genomic screening in enhancing drug efficacy.