Targeting aberrant signaling pathways: novel therapeutic approaches for the treatment of acute leukemia

Over the last decades, a significant progress in the understanding of leukemogenesis has been achieved highlighting several of the molecular alterations responsible for leukemia cell proliferation and survival. Despite novel findings, advances in treatment of acute myeloid and lymphoblastic leukemia (AML, ALL) have been quite modest with the prognosis of patients remaining, generally, severe. Therefore, investigation has been prompted towards new targeted approaches focused on the aberrant networks that sustain blast proliferation, survival and drug resistance. Phosphatidyl-inositol-3 kinases (PI3Ks) belong to a family of evolutionary conserved enzymes that regulates cell omeostasis by transducing signals from the microenvironment to the nucleus through protein kinases such as Akt and the mammalian target of rapamycin (mTOR). Deregulation of the PI3K/Akt/mTOR signaling has been associated with several human cancers, and its constitutive activation is frequently found in AML patients contributing to chemoresistance, disease progression and unfavorable outcome. Over the years, several small molecules that target the PI3K/Akt/mTOR signaling has been investigated showing potential therapeutic efficacy in AML alone and in combination with chemotherapeutic drugs. Defects in apoptosis signaling which result in evasion from programmed cell death are hallmarks of cancer cells. The Bcl-2 family comprises highly conserved proteins involved in the regulation of apoptosis. Alterations in the expression levels and/or function of Bcl-2 members are widely observed in human neoplasia, including ALL, allowing cancer cells to survive irrespective of physiological or drug-induced death signals. An efficient targeting of the aberrant apoptotic machinery by BH3 mimetics has been recognized as an important strategy to improve actual ALL therapy. However, pre-existing or acquired resistance mechanisms represent a considerable challenge for the clinical use of these molecules. In this study, two novel therapeutic approaches based on target inhibition of different aberrant signaling pathways in AML and ALL have been investigated. The first study evaluates the pre-clinical activity of NVP-BKM120 (cited hereafter as BKM120), a novel selective pan-class I PI3K inhibitor, on AML cells. Previous reports have shown that BKM120 exerted cytotoxic effects in vitro and in vivo on several solid tumors and on some hematological malignancies through the selective inhibition of PI3K/Akt/mTOR activity. Conversely, a limited cytotoxicity was observed towards normal cells. Moreover, this compound synergized with well-established chemotherapeutic drugs (i.e. doxorubicin, vincristine) and novel small molecules (i.e. Mek, Bcl-2 and mTOR inhibitors). Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling at low micromolar concentrations, promoting cell growth arrest and a significant apoptosis induction in a dose- and time-dependent manner in AML cells. Conversely, no relevant cytotoxicity is observed on resting or PHA-activated normal mononuclear cells isolated from healthy volunteers. BKM120-induced cytotoxicity is associated with a profound modulation of the metabolic behavior in both cell models and primary samples, particularly reducing the oxidative capabilities and the glycolytic rates of AML cells. In addition, BKM120 synergizes with the glycolytic inhibitor dichloroacetate (DCA), triggering massive apoptosis at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibits leukemia progression and improves the overall survival of treated mice without inducing any detectble side effects. In the second study, a novel combined approach to overcome ABT-737 acquired resistance in ALL with the simultaneous inhibition of mTOR pathway by CCI-779 has been explored. It has been shown that activated PI3K/Akt/mTOR pathway decrease ABT-737 effectiveness in cancer cells by upregulating anti-apoptotic Bcl-2 family members (i.e. Mcl-1). Consistently, simultaneous inhibition strongly potentiated cytotoxicity of single agents while exerted only modest toxicity toward normal progenitors in vitro and in vivo. This strategy also triggered apoptosis under hypoxic conditions, thus indicating a potential efficacy even within tumor microenvironment. We demonstrate that co-targeting of Bcl-2 and mTOR strongly sensitized ALL cells to apoptosis, reverting the acquired ABT-737 resistance of cell models and primary samples. Analysis of signaling modulations in newly sensitized ABT-737 resistant cells reveals that ABT-737 plus CCI-779 combination reduces 4EBP1 activation and Mcl-1expression. Since the involvement of proteasomal degradation was excluded, inhibition of 4EBP1-dependent protein translation may be responsible for the synergistic interaction between ABT-737 and CCI-779. However, in some ALL models, Mcl-1 may be not the unique determinant of ABT-737 resistance since knockdown of protein expression by RNA interference does not result in significant changes of cytotoxicity. Taken together, our results (i) document that BKM120, as single agent or in combination with glycolytic modulators, has a significant anti-leukemic activity towards AML cells, thus supporting its clinical evaluation as a novel targeted agent for this hematological malignancy and (ii) identify a novel therapeutic approach to overcome ABT-737 resistance in ALL, demonstrating the synergistic interaction with the mTOR inhibitor CCI-779 and further clarify the role of Mcl-1 in mediating ABT-737 resistance.