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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.

Simple Summary Targeting aberrant DNA repair in cancers in addition to transcription and replication is an area of interest for cancer researchers. Inhibition of DNA repair selectively in cancer cells leads to cytotoxic or cytostatic effects and overcomes survival advantages imparted by chromosomal translocations or mutations. In this review, we highlight the relevance of DNA repair-linked events in developmental diseases and cancers and also discuss mechanisms to overcome these events that participate in different cellular processes. Abstract Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

// Danielle P. Johnson 2,* , Gabriella S. Spitz 1,* , Shweta Tharkar 1 , Steven N. Quayle 3 , Jeffrey R. Shearstone 3 , Simon Jones 3 , Maria E. McDowell 1 , Hannah Wellman 1 , Jessica K. Tyler 4 , Bradley R. Cairns 2 , Mahesh B. Chandrasekharan 1 and Srividya Bhaskara 1,2 1 Department of Radiation Oncology, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 2 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, UT 3 Acetylon Pharmaceuticals, Inc., Boston, MA 4 Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX * These authors contributed equally to the study Correspondence: Srividya Bhaskara, email: // Keywords : epigenetics, DNA repair, HDAC1,2, chromatin, EZH2 Received : November 01, 2014 Accepted : December 28, 2014 Published : December 31, 2014 Abstract Gain-of-function mutations in the catalytic site of EZH2 (Enhancer of Zeste Homologue 2), is observed in about 22% of diffuse large B-cell lymphoma (DLBCL) cases. Here we show that selective inhibition of histone deacetylase 1,2 (HDAC1,2) activity using a small molecule inhibitor causes cytotoxic or cytostatic effects in EZH2 gain-of-function mutant (EZH2 GOF ) DLBCL cells. Our results show that blocking the activity of HDAC1,2 increases global H3K27ac without causing a concomitant global decrease in H3K27me3 levels. Our data shows that inhibition of HDAC1,2 is sufficient to decrease H3K27me3 present at DSBs, decrease DSB repair and activate the DNA damage response in these cells. In addition to increased H3K27me3, we found that the EZH2 GOF DLBCL cells overexpress another chemotherapy resistance factor − B-lymphoma and BAL-associated protein (BBAP). BBAP monoubiquitinates histone H4K91, a residue that is also subjected to acetylation. Our results show that selective inhibition of HDAC1,2 increases H4K91ac, decreases BBAP-mediated H4K91 monoubiquitination, impairs BBAP-dependent DSB repair and sensitizes the refractory EZH2 GOF DLBCL cells to treatment with doxorubicin, a chemotherapy agent. Hence, selective HDAC1,2 inhibition provides a novel DNA repair mechanism-based therapeutic approach as it can overcome both EZH2- and BBAP-mediated DSB repair in the EZH2 GOF DLBCL cells.

The Achilles heel of conventional and targeted anticancer treatments is intrinsic or acquired resistance following Darwinian selection, i.e. treatment toxicity place the surviving cells under intense evolutionary selective pressure to develop resistance. Here, we demonstrate that AsiDNA, the first antitumor drug with an agonist activity, can instead drive the evolution of malignant cells toward a different trajectory. Specifically, long term exposure of cancer cells to the strong alarm signal, generated by the AsiDNATM DNA repair inhibitors, did not promote resistance emergence. It induces a stable new state characterized by the down regulation of the targeted pathways that persists for months after treatments. This property is due to the original mechanism of action of AsiDNATM, which acts by over-activating a “false” signaling of DNA damage through DNA-PK and PARP enzymes, and is not observed with other DNA repair inhibitors such as the PARP inhibitors olaparib and talazoparib. Transcriptomic analysis of AsiDNATM treated independent populations derived from lung and breast cancers revealed the common evolution of independent AsiDNA-treated samples, resulting in a general down-regulation of enzymes involved in DNA repair and oncogenesis, and a specific down regulation of DNA-PK targets. Similar down regulation of DNA-PK targets was observed in xenografted tumor in animal models after three cycles of AsiDNA treatment. Chromatin analysis revealed a major change in AsiDNATM treated populations with an increased nucleosome stability that could be responsible for the epigenetic modification observed. Long term treatment with AsiDNATM induces a new “alarm down” state in the tumor cells that increase its efficacy. These results suggest that agonist drugs such as AsiDNATM could promote a state-dependent tumor cell evolution by lowering their ability to respond to damage signal as predicted by the ecological and evolutionary “smoke detector principle”. Citation Format: Wael Jdey, Maria Kozlac, Sergey Alekseev, Danielle Johnson, françoise Bono, Srividya bhaskara, frederic thomas, Marie Dutreix. AsiDNATM, a targeted therapy with no acquired resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2095.

Introduction: The Philadelphia (Ph) chromosome was the first reported chromosomal rearrangement linked to a human malignancy. Ph-positive precursor B-cell acute lymphoblastic leukemic (Ph+Pre-B-ALL) expressing the BCR-ABL1 oncoprotein is a major subclass of ALL with poor prognosis and high relapse rates in adults. BCR-ABL1 drives an aberrantly hyperactive DNA repair that confers survival advantages to leukemic cells and serves as its ‘Achilles' heel'. We used HDAC1,2 selective inhibitor in isolation or in combination with doxorubicin (a chemotherapy regimen component used for treating pre-B-ALL) to combat the aberrant DSB repair processes. Experimental design: We used a combination of molecular and cell biology, proteomics and functional genomics-based approaches and primary patient-derived xenograft mouse models to understand the genome stability mechanisms targeted by HDAC1,2 inhibition to overcome oncogenic signals promoted by the BCR-ABL1 fusion protein. Results: Mass-spectrometry analysis and DNA repair assays showed that HDAC1,2 inhibition and doxorubicin treatments impact common as well as distinct DNA repair and genome maintenance networks including decreased chromatin association of Mre11-Rad51-DNA ligase 1, the central hub of DNA repair. Translating this mechanistic knowledge, we then showed that the HDAC1,2 inhibitor either alone or when combined with a low dose doxorubicin caused cytotoxicity in primary patient Ph+ Pre-B-ALL cells, and decreased leukemia burden without adverse side-effects in vivo in refractory Ph+ Pre-B-ALL PDX models that we created. We recently found that HDAC1,2 targets H4K91ac mark in Ph+Pre-B-ALL cells. H4K91ac occurs within the body of nucleosome core and thus directly controls nucleosome or chromatin integrity. Increased H4K91ac following HDAC1,2 inhibition occurs around DSB sites genome-wide could cause a delay in the rate at which DSBs are repaired in Ph+Pre-B-ALL cells as a result of defective nucleosome structure around DSB sites. Conclusion: Our comprehensive study using a multipronged approach showed that HDAC1,2 inhibition as a monotherapy or in combination with a low-dose doxorubicin can override DSB repair ‘addiction' in Ph+ Pre-B-ALL. We are currently using the genome-wide ChIP-seq approach and an homing endonuclease DSB reporter system to examine how HDAC1,2 inhibition changes the chromatin landscape in Ph+Pre-B-ALL cells. Citation Format: Danielle Johnson, Shwetha Tharkar, Simon Jones, Matt Jarpe, Mahesh Chandrasekharan, Srividya Bhaskara. Targeting DNA repair in Philadelphia positive B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5844.