Inhibition of Rac GTPase signaling and downstream prosurvival Bcl-2 proteins as combination targeted therapy in MLL-AF9 leukemia

Abstract

The Rac family of small Rho GTPases coordinates diverse cellular functions in hematopoietic cells including adhesion, migration, cytoskeleton rearrangements, gene transcription, proliferation, and survival. The integrity of Rac signaling has also been found to critically regulate cellular functions in the initiation and maintenance of hematopoietic malignancies. Using an in vivo gene targeting approach, we demonstrate that Rac2, but not Rac1, is critical to the initiation of acute myeloid leukemia in a retroviral expression model of MLL-AF9 leukemogenesis. However, loss of either Rac1 or Rac2 is sufficient to impair survival and growth of the transformed MLL-AF9 leukemia. Rac2 is known to positively regulate expression of Bcl-2 family proteins toward a prosurvival balance. We demonstrate that disruption of downstream survival signaling through antiapoptotic Bcl-2 proteins is implicated in mediating the effects of Rac2 deficiency in MLL-AF9 leukemia. Indeed, overexpression of Bcl-xL is able to rescue the effects of Rac2 deficiency and MLL-AF9 cells are exquisitely sensitive to direct inhibition of Bcl-2 family proteins by the BH3-mimetic, ABT-737. Furthermore, concurrent exposure to NSC23766, a smallmolecule inhibitor of Rac activation, increases the apoptotic effect of ABT-737, indicating the Rac/Bcl-2 survival pathway may be targeted synergistically. (Blood. 2011;118(19):5235-5245) IntroductionThe pathway to aberrant self-renewal and survival in acute myeloid leukemia (AML) commonly begins with specific chromosomal lesions. In a recent study, rearrangement of the mixed-lineageleukemia (MLL) gene on chromosome 11q23 was the most prevalent chromosomal abnormality in pediatric AML, constituting 16% of all patients. 1 The most common MLL gene rearrangement, seen in 49% of all MLL-positive patients, involved a balanced translocation with the AF9 gene on chromosome 9, t(9;11)(p21-22; q23), producing the fusion protein MLL-AF9 (MA9). 1 AML with the MA9 fusion displays a myelomonocytic phenotype and is associated with intermediate prognostic risk. AF9 is one of several MLL fusion partners found to recruit a macromolecular complex involving positive transcription elongation factor B (pTEFb) and the histone methyltransferase DOT1L to the fusion protein with preserved DNA-binding domains at the MLL amino terminus. 2,3 This pTEFb/DOT1L complex results in aberrant transcription elongation and histone H3K79 methylation of MLL target genes leading to their overexpression. [4][5][6][7] Genetic, epigenetic, and signaling changes accumulate leading to leukemic transformation. Using retroviral vectors to express MA9 in hematopoietic stem and progenitor cells (HSC/Ps), we and others have been able to model the initiation and progression of AML in both xenograft and pure murine genetic models. [8][9][10][11][12] Rac GTPases have garnered interest as a therapeutic target in hematologic malignancies because of their central role in coordinating diverse cellular functions in response to stimuli within the cell microenvironment. [13][1...

Tiam1/Rac1 signals contribute to the proliferation and chemoresistance, but not motility, of chronic lymphocytic leukemia cells

“…56 Concurrent exposure to NSC-23766 increased the apoptotic effect of the Bcl-2 inhibitor and BH3 mimetic ABT-737 in leukemia cells. 57 Bcl-2 overexpression is a hallmark of CLL 58 . Coherently, BH3 mimetics have proven to be efficient in patients with CLL, but with heterogeneity, 59 and in microenvironmentmediated drug resistance.…”

Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells

“…The MLL fusion protein MLL-AF9 immortalizes CB cells in vitro and produces human leukemia in immunodeficient mice (20). These engineered pre-leukemic and leukemic cells recapitulate many features of the clinical diseases and have been useful in testing different therapeutic strategies (21)(22)(23)(24)(25).…”

“…The dysregulated genes in Runx1/Cbfb-deleted cells contained several known RUNX1 targets, including RUNX1 itself and C/EBPα (Supplemental Tables 3 and 4). Among these candidate Runx target genes, we focused on Bcl2, which was shown to be important for the pathogenesis of MLL fusion leukemia (22,36). It was also shown that RUNX1 binds to the TGTGGT sequence in the BCL2 promoter (37).…”

Frat2 mediates the oncogenic activation of Rac by MLL fusions

“…Recent studies have demonstrated that progression of preleukemic to leukemic MLL fusion cells involves increased canonical Wnt signaling 16 and Rac GTPase activation 17 ; both of these signaling pathways play critical roles in leukemia progression. 10,16,[18][19][20] The sensitivity of MLL-rearranged AML cells to Rac inhibition suggests that targeting Rac activity or signaling pathways leading to Rac activation may represent novel therapeutic opportunities. In support of this, pharmacologic Rac inhibition was found to interfere with leukemic engraftment of MLL-rearranged human AML cell lines.…”

“…57,58 In MLL-rearranged AML, the importance of Rac activation to the leukemogenic function of MLL fusions has also been recently demonstrated. 10,19,20 By identifying the mediators of MLL fusioninduced Rac activation, the present study offers new candidates for novel therapeutic targeting. The inhibition of leukemic progression and Rac activation by FRATN suggests that targeting the FRAT2/ DVL/GSK3␤ axis would be an effective means of interfering with MLL fusion activity.…”