Duvelisib, an oral dual inhibitor of PI3K-δ and PI3K-γ, is in phase III trials for the treatment of chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin’s lymphoma (iNHL). In CLL, duvelisib monotherapy is associated with high iwCLL and nodal response rates, but complete remissions are rare. To characterize the molecular effect of duvelisib, we obtained samples from CLL patients on the duvelisib phase I trial. Gene-expression studies (RNA seq, Nanostring, Affymetrix array, and real time RT-PCR) demonstrated increased expression of BCL2 along with several BH3-only pro-apoptotic genes. In concert with induction of transcript levels, reverse phase protein arrays and immunoblots confirmed increase at the protein level. The BCL2 inhibitor venetoclax induced greater apoptosis in ex-vivo cultured CLL cells obtained from patients on duvelisib compared to pre-treatment CLL cells from the same patients. In vitro combination of duvelisib and venetoclax resulted in enhanced apoptosis even in CLL cells cultured under conditions that simulate the tumor microenvironment. These data provide a mechanistic rationale for testing the combination of duvelisib and venetoclax in the clinic. Such combination regimen (NCT02640833) is being evaluated for patients with B-cell malignancies including CLL.

IntroductionStudies of genes associated with retrovirus-induced neoplasia form the basis of much of our present knowledge of oncogenes, and have contributed to our understanding of both gene function and the neoplastic process. 1 Friend murine leukemia virus (F-MuLV) does not contain any oncogenic sequences. Instead it induces tumors by activating or inactivating oncogenes or tumor suppressor genes, respectively, through a process known as retroviral insertional mutagenesis. 2 Previous studies in our laboratory and others have demonstrated that the pivotal genetic event associated with erythroleukemia initiation is the insertional activation of the Ets-related gene Fli-1. [3][4][5] Progression toward more malignant stages has been shown to be associated with insertional inactivation of either p53 or p45 NFE2. [6][7][8][9][10] To further investigate the role of p53 in F-MuLV-induced erythroleukemia, p53-deficient mice were infected with F-MuLV. While the loss of p53 in primary erythroleukemias accelerated tumor initiation and immortalization, 11 10% of these tumors did not acquire an insertional activation of the Fli-1 locus, and the cell lines established from these erythroleukemias did not express endogenous Fli-1. The transformation of these cells is attributable to insertional activation or inactivation of another gene, since F-MuLV-induced erythroleukemia usually requires Fli-1 activation for erythroid transformation. 3 Therefore, the investigation of a novel site of proviral integration is necessary to uncover the mechanism of transformation in the Fli-1-negative erythroleukemias. This study describes the identification of a novel integration site, designated Friend murine leukemia integration site 3 (Fli-3), containing sequences identical to the miRNA gene family.miRNAs are small, non-protein-encoding RNAs that play important roles in a variety of biologic processes. They act by binding to complementary sequences in the 3Ј untranslated region (UTR) of the mRNA of their target gene. This binding process reduces the stability of these mRNAs, thereby altering the protein expression. 12,13 Some miRNA genes exist in clusters that can be expressed as single transcriptional polycistron. 14 Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of human cancers. [15][16][17][18][19][20] Fli-3 is a murine homologue of the human C13orf25 gene, a newly identified target gene for 13q31-q32 chromosomal amplification in B-cell-type lymphomas. 21 C13orf25 contains sequences corresponding to the mir-17-92 polycistron. He et al 22 have recently reported that mir-17-92 is highly expressed in B-cell lymphomas and its expression in c-myc-overexpressing transgenic mice accelerated tumorigenicity by an as-yet-undefined process. Hayashita et al 18 also showed that mir-17-92 is overexpressed in human lung cancer and enhances cell proliferation. Interestingly, the Fli-3 transcript contains the identical mir-17-92 sequence, suggesting that this murine homologue may play a similar rol...

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Survival of CLL cells due to the presence of Bcl-2 and Mcl-1 has been established. Direct inhibition of Bcl-2 by venetoclax and indirect targeting of Mcl-1 with transcription inhibitors have been successful approaches for CLL. AMG-176 is a selective and direct antagonist of Mcl-1, which has shown efficacy in several hematologic malignancies; however, its effect on CLL is elusive. We evaluated biological and molecular effects of AMG-176 in primary CLL cells.Experimental Design: Using samples from patients (n ¼ 74) with CLL, we tested effects of AMG-176 on CLL and normal hematopoietic cell death and compared importance of CLL prognostic factors on this biological activity. We evaluated CLL cell apoptosis in the presence of stromal cells and identified cell death pathway including stabilization of Mcl-1 protein. Finally, we tested a couplet of AMG-176 and venetoclax in CLL lymphocytes.Results: AMG-176 incubations resulted in time-and dosedependent CLL cell death. At 100 and 300 nmol/L, there was 30% and 45% cell death at 24 hours. These concentrations did not result in significant cell death in normal hematopoietic cells. Presence of stroma did not affect AMG-176-induced CLL cell death. IGHV unmutated status, high b2M and Mcl-1 protein levels resulted in slightly lower cell death. Mcl-1, but not Bcl-2 protein levels, in CLL cells increased with AMG-176. Low concentrations of venetoclax (1-30 nmol/L) were additive or synergistic with AMG-176.Conclusions: AMG-176 is active in inducing CLL cell death while sparing normal blood cells. Combination with low-dose venetoclax was additive or synergistic.