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.

AATF mediates an antiapoptotic effect of the unfolded protein response through transcriptional regulation of AKT1.

Endoplasmic reticulum (ER) stress-mediated cell death has an important role in the pathogenesis of chronic diseases, including diabetes and neurodegeneration. Although proapoptotic programs activated by ER stress have been extensively studied, identification and characterization of antiapoptotic programs that counteract ER stress are currently incomplete. Through the gene expression profiling of beta-cells lacking Wolfram syndrome 1 gene (WFS1), a causative gene for Wolfram syndrome, we discovered a novel antiapoptotic gene of the unfolded protein response (UPR), apoptosis antagonizing transcription factor (AATF). Here, we study the regulation of AATF, identify its target genes, and determine the basis for its antiapoptotic activities in response to ER stress. We show that AATF is induced by ER stress through the PERK-eIF2alpha pathway and transcriptionally activates the v-akt murine thymoma viral oncogene homolog 1 (AKT1) gene through signal transducer and activator of transcription 3 (Stat3), which sustains Akt1 activation and promotes cell survival. Ectopic expression of AATF or a constitutively active form of AKT1 confers on cells resistance to ER stress-mediated cell death, whereas RNAi-mediated knockdown of AATF or AKT1 renders cells sensitive to ER stress. We also discovered a positive crosstalk between the AATF and WFS1 signaling pathways. Thus, WFS1 deficiency or AATF deficiency mediates a self-perpetuating cycle of cell death. Our results reveal a novel antiapoptotic program relevant to the treatment of diseases caused by ER stress-mediated cell death.

Biochemical characterization of the small heat shock protein IbpB from Escherichia coli.

Escherichia coli IbpB was overexpressed in a strain carrying a deletion in the chromosomal ibp operon and purified by refolding. Under our experimental conditions, IbpB exhibited pronounced size heterogeneity. Basic oligomers, roughly spherical and approximately 15 nm in diameter, interacted to form larger particles in the 100-200-nm range, which themselves associated to yield loose aggregates of micrometer size. IbpB suppressed the thermal aggregation of model proteins in a concentration-dependent manner, and its CD spectrum was consistent with a mostly beta-pleated secondary structure. Incubation at high temperatures led to a partial loss of secondary structure, the progressive exposure of tryptophan residues to the solvent, the dissociation of high molecular mass aggregates into approximately 600-kDa oligomers, and an increase in surface hydrophobicity. Structural changes were reversible between 37 and 55 degrees C, and, up to 55 degrees C, hydrophobic sites were reburied upon cooling. IbpB exhibited a biphasic unfolding trend upon guanidine hydrochloride (GdnHCl) treatment and underwent comparable conformational changes upon melting and during the first GdnHCl-induced transition. However, hydrophobicity decreased with increasing GdnHCl concentrations, suggesting that efficient exposure of structured hydrophobic sites involves denaturant-sensitive structural features. By contrast, IbpB hydrophobicity rose at high NaCl concentrations and increased further at high temperatures. Our results support a model in which temperature-driven conformational changes lead to the reversible exposure of normally shielded binding sites for nonnative proteins and suggest that both hydrophobicity and charge context may determine substrate binding to IbpB.