HDAC1,2 inhibition and doxorubicin impair Mre11-dependent DNA repair and DISC to override BCR-ABL1-driven DSB repair in Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph+) B-cell precursor acute lymphoblastic leukemia (ALL) expressing BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis. BCR-ABL1-expressing leukemic cells are highly dependent on double-strand break (DSB) repair signals for their survival. Here we report that a first-in-class HDAC1,2 selective inhibitor and doxorubicin (a hyper-CVAD chemotherapy regimen component) impair DSB repair networks in Ph+ B-cell precursor ALL cells using common as well as distinct mechanisms. The HDAC1,2 inhibitor but not doxorubicin alters nucleosomal occupancy to impact chromatin structure, as revealed by MNase-Seq. Quantitative mass spectrometry of the chromatin proteome along with functional assays showed that the HDAC1,2 inhibitor and doxorubicin either alone or in combination impair the central hub of DNA repair, the Mre11-Rad51-DNA ligase 1 axis, involved in BCR-ABL1-specific DSB repair signaling in Ph+ B-cell precursor ALL cells. HDAC1,2 inhibitor and doxorubicin interfere with DISC (DNA damage-induced transcriptional silencing in cis)) or transcriptional silencing program in cis around DSB sites via chromatin remodeler-dependent and -independent mechanisms, respectively, to further impair DSB repair. HDAC1,2 inhibitor either alone or when combined with doxorubicin decreases leukemia burden in vivo in refractory Ph+ B-cell precursor ALL patient-derived xenograft mouse models. Overall, our novel mechanistic and preclinical studies together demonstrate that HDAC1,2 selective inhibition can overcome DSB repair 'addiction' and provide an effective therapeutic option for Ph+ B-cell precursor ALL.

Regulation of PI3K signaling in T-cell acute lymphoblastic leukemia: a novel PTEN/Ikaros/miR-26b mechanism reveals a critical targetable role for PIK3CD.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy, and T-ALL patients are prone to early disease relapse and suffer from poor outcomes. The PTEN, PI3K/AKT and Notch pathways are frequently altered in T-ALL. PTEN is a tumor suppressor that inactivates the PI3K pathway. We profiled miRNAs in Pten-deficient mouse T-ALL and identified miR-26b as a potentially dysregulated gene. We validated decreased expression levels of miR-26b in mouse and human T-ALL cells. In addition, expression of exogenous miR-26b reduced proliferation and promoted apoptosis of T-ALL cells in vitro, and hindered progression of T-ALL in vivo. Furthermore, miR-26b inhibited the PI3K/AKT pathway by directly targeting PIK3CD, the gene encoding PI3Kδ, in human T-ALL cell lines. ShRNA for PIK3CD and CAL-101, a PIK3CD inhibitor, reduced the growth and increased apoptosis of T-ALL cells. Finally, we showed that PTEN induced miR-26b expression by regulating the differential expression of Ikaros isoforms that are transcriptional regulators of miR-26b. These results suggest that miR-26b functions as a tumor suppressor in the development of T-ALL. Further characterization of targets and regulators of miR-26b may be promising for the development of novel therapies.

Multiple receptor tyrosine kinases regulate HIF-1alpha and HIF-2alpha in normoxia and hypoxia in neuroblastoma: implications for antiangiogenic mechanisms of multikinase inhibitors.

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1alpha). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1alpha levels and whether RTKs can regulate HIF-2alpha as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-alpha and -beta. Ligand-induced activation of multiple RTKs upregulated HIF-1alpha levels, whereas activation of VEGFR-1 alone upregulated HIF-2alpha. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1alpha and HIF-2alpha through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1alpha levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1alpha and HIF-2alpha.

Regulation of NOTCH signaling by reciprocal inhibition of HES1 and Deltex 1 and its role in osteosarcoma invasiveness.

The highly conserved NOTCH signaling pathway has many essential functions in the development of diverse cells, tissues and organs from Drosophila to humans, and dysregulated NOTCH signaling contributes to several disorders, including vascular and bone defects, as well as several cancers. Here we describe a novel mechanism of NOTCH regulation by reciprocal inhibition of two NOTCH downstream effectors: Deltex1 and HES1. This mechanism appears to regulate invasion of osteosarcoma cells, as Deltex1 blocks osteosarcoma invasiveness by downregulating NOTCH/HES1 signaling. The inhibitory effect of endogenous Deltex1 on NOTCH signaling is mediated through binding with the intracellular domain of NOTCH and ubiquitination and degradation of NOTCH receptors. Conversely, we show that the NOTCH target gene HES1 causes transcriptional inhibition of Deltex1 by directly binding to the promoter of Deltex1. An HES1 binding site is identified 400 bp upstream of the transcription start site of Deltex1. HES1-mediated repression of Deltex1 requires the C-terminal H3/H4 and WRPW domains of HES1, which associate with the TLE/Groucho corepressors. Taken together, we define a molecular mechanism regulating NOTCH signaling by reciprocal inhibition of the NOTCH target genes HES1 and Deltex1 in mammalian cells. This mechanism may have important clinical implications for targeting NOTCH signaling in osteosarcoma and other cancers.

The Gfi-1 proto-oncoprotein contains a novel transcriptional repressor domain, SNAG, and inhibits G1 arrest induced by interleukin-2 withdrawal.

The Gfi-1 proto-oncogene is activated by provirus insertion in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced thymomas and encodes a nuclear, sequence-specific DNA-binding protein. Here we show that Gfi-1 is a position- and orientation-independent active transcriptional repressor, whose activity depends on a 20-amino-acid N-terminal repressor domain, coincident with a nuclear localization motif. The sequence of the Gfi-1 repressor domain is related to the sequence of the repressor domain of Gfi-1B, a Gfi-1-related protein, and to sequences at the N termini of the insulinoma-associated protein, IA-1, the homeobox protein Gsh-1, and the vertebrate but not the Drosophila members of the Snail-Slug protein family (Snail/Gfi-1, SNAG domain). Although not functionally characterized, these SNAG-related sequences are also likely to mediate transcriptional repression. Therefore, the Gfi-1 SNAG domain may be the prototype of a novel family of evolutionarily conserved repressor domains that operate in multiple cell lineages. Gfi-1 overexpression in IL-2-dependent T-cell lines allows the cells to escape from the G1 arrest induced by IL-2 withdrawal. Since a single point mutation in the SNAG domain (P2A) inhibits both the Gfi-1-mediated transcriptional repression and the G1 arrest induced by IL-2 starvation, we conclude that the latter depends on the repressor activity of the SNAG domain. Induction of Gfi-1 may therefore contribute to T-cell activation and tumor progression by repressing the expression of genes that inhibit cellular proliferation.

Gfi-1 encodes a nuclear zinc finger protein that binds DNA and functions as a transcriptional repressor.

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence-specific manner. The Gfi-1 binding site, TAAATCAC(A/T)GCA, was defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oliginucleotides defined the relative importance of specific bases at the consensus binding site. Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding. Potential Gfi-1 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression was abrogated by mutation of critical residues in the two Gfi-1 binding sites. These results suggest that Gfi-1 may play a role in HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.