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Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98 fusion proteins. Abl1−/− murine hematopoietic cells transduced with AML1-ETO and NUP98-PMX1 gained proliferation advantage when compared to Abl1 + /+ counterparts. Conversely, overexpression and pharmacological stimulation of ABL1 kinase resulted in reduced proliferation. To pinpoint mechanisms facilitating the transformation of ABL1-deficient cells, Abl1 was knocked down in 32Dcl3-Abl1ko cells by CRISPR/Cas9 followed by the challenge of growth factor withdrawal. 32Dcl3-Abl1ko cells but not 32Dcl3-Abl1wt cells generated growth factor-independent clones. RNA-seq implicated PI3K signaling as one of the dominant mechanisms contributing to growth factor independence in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib exerted selective activity against Lin-cKit+ NUP98-PMX1;Abl1−/− cells when compared to the Abl1 + /+ counterparts. Since the role of ABL1 in DNA damage response (DDR) is well established, we also tested the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi). AML1-ETO;Abl1−/− and NUP98-PMX1;Abl1−/− cells were hypersensitive to DNA-PKi and ATRi, respectively, when compared to Abl1 + /+ counterparts. Moreover, ABL1 kinase inhibitor enhanced the sensitivity to PI3K, DNA-PKcs and ATR inhibitors. In conclusion, we showed that ABL1 kinase plays a tumor suppressor role in hematological malignancies induced by AML1-ETO and NUP98-PMX1 and modulates the response to PI3K and/or DDR inhibitors.

Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/one-carbon cycle metabolism contributed to accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1]. To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLq) by ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLq and its proteasomal degradation. Overexpression of POLq in OTK-positive cells resulted in efficient repair of DPC-containing DNA double-strand breaks (DSBs) by POLq-mediated end-joining (TMEJ). Transforming activity of OTKs and other leukemia-inducing oncogenes, especially of those causing inhibition of BRCA1/2 -mediated homologous recombination (HR) with and without concomitant inhibition of DNA-PK -dependent non-homologous end-joining (D-NHEJ), was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLq polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitor or DPC-inducing drug etoposide enhanced anti-leukemia effect of POLq inhibitor (POLqi) in vitro and in vivo. In conclusion, we demonstrated that POLq plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde and that it can be targeted to achieve therapeutic effect.

Despite much of the past 2 years being engulfed by the devastating consequences of the SAR-CoV-2 pandemic, significant progress, even breathtaking, occurred in the field of chronic myeloid malignancies. Some of this was show-cased at the 15th Post-American Society of Hematology (ASH) and the 25th John Goldman workshops on myeloproliferative neoplasms (MPN) held on 9th-10th December 2020 and 7th-10th October 2021, respectively. The inaugural Post-ASH MPN workshop was set out in 2006 by John Goldman (deceased) and Tariq Mughal to answer emerging translational hematology and therapeutics of patients with these malignancies. Rather than present a resume of the discussions, this perspective focuses on some of the pivotal translational hematology and therapeutic insights in these diseases.

Key Points c-KIT activating mutations cause resistance to PARP inhibitor in AML1-ETO–positive leukemias. c-KIT inhibitor avapritinib downregulates BRCA1/2 and DNA-PK catalytic subunit to restore the sensitivity to PARP inhibitor.

Despite recent progress in treatment, prognostication for T-cell acute lymphoblastic leukemia (T-ALL) patients, including those with early T-cell precursor ALL (ETP-ALL), is rather poor [1]. Theref...

// Kaushiki Mukherjee 1,* , Xiaojin Sha 1,* , Andrew Magimaidas 1,3,* , Silvia Maifrede 1,2 , Tomasz Skorski 1,2 , Ravi Bhatia 4 , Barbara Hoffman 1,5 and Dan A. Liebermann 1,5 1 Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA 2 Department of Microbiology and Immunology, Temple University, Philadelphia, PA, USA 3 Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4 Division of Hematology and Oncology, University of Alabama, Tuscaloosa, AL, USA 5 Department of Medical Genetics and Molecular Biochemistry, Temple University, Philadelphia, PA, USA * These authors have contributed equally to this work Correspondence to: Dan A. Liebermann, email: // Keywords : Gadd45a, chronic myelogenous leukemia, stress response protein, tumor suppressor Received : September 23, 2016 Accepted : November 23, 2016 Published : January 10, 2017 Abstract The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of BCR-ABL1 tyrosine kinase - positive chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" the disease in majority of patients because CML-CP cells are elusive targets even for the most advanced therapies employing second and third generation of TKIs. Therefore, new treatment modalities are necessary to improve therapeutic outcomes. We showed before that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. The aim of the study was to test whether simultaneous inhibition of signaling pathways activated by BCR-ABL1 and PAK kinases may improve the treatment of CML. Special attention was focused on PAK1 and PAK2, which are expressed in hematopoietic cells and can play an important role in the promotion of CML cells proliferation and survival. PAK kinases were targeted by small molecule inhibitor IPA-3 (inhibitor of PAK1) and shRNA construct for PAK2, BCR-ABL1 kinase was inhibited by imatinib. The studies were carried out using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed CML-CP patients and healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) BCR-ABL1-transformed 32Dcl3 cell line cells. We show here that inhibition of PAK1 or/and PAK2 kinases activity enhanced the effect of IM against CML cells without affecting normal counterparts. We observed that the combined use of IM with IPA-3 increased growth inhibition and apoptosis of leukemia cells. To evaluate the type of drugs interaction median effect analysis method was used. The studies revealed that the type and strength of drug interaction depend on drug concentration. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Altogether, we postulate that BCR-ABL1 kinase inhibitor should be combined with PAK1/2 inhibitor to facilitate eradication of CML cells. Disclosures No relevant conflicts of interest to declare.

Philadelphia negative (Ph-neg) myeloproliferative neoplasms (MPN) are a heterogenous group of clonal stem cell disorders. Approved treatment options include hydroxyurea, anagrelide, and ruxolitinib, which are not curative. The concept of synthetic lethality may become an additional therapeutic strategy in these diseases. In our study, we show that DNA repair is altered in classical Ph-neg MPN, as analyzed by gene expression analysis of 11 genes involved in the homologous recombination repair pathway (HRR), the non-homologous end-joining pathway (NHEJ), and the single-strand break repair pathway (SSB). Altogether, peripheral blood-derived cells from 57 patients with classical Ph-neg MPN and 13 healthy controls were analyzed. LIG3 as an essential part of the SSB was significantly lower expressed compared to controls in all three entities (essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF)). In addition, while genes of other DNA-repair pathways showed-possibly compensatory-increased expression in ET (HRR, NHEJ) and PV (NHEJ), MF samples displayed downregulation of all genes involved in NHEJ. With regard to the JAK2 mutational status (analyzed in ET and MF only), no upregulation of the HRR was detected. Though further studies are needed, based on these findings, we conclude that synthetic lethality may become a promising strategy in treating patients with Ph-neg MPN.

The transcription factor JUN is frequently overexpressed in multiple genetic subtypes of acute myeloid leukemia (AML); however, the functional role of JUN in AML is not well defined. Here we report that short hairpin RNA (shRNA)-mediated inhibition of JUN decreases AML cell survival and propagation in vivo. By performing RNA sequencing analysis, we discovered that JUN inhibition reduces the transcriptional output of the unfolded protein response (UPR), an intracellular signaling transduction network activated by endoplasmic reticulum (ER) stress. Specifically, we found that JUN is activated by MEK signaling in response to ER stress, and that JUN binds to the promoters of several key UPR effectors, such as XBP1 and ATF4, to activate their transcription and allow AML cells to properly negotiate ER stress. In addition, we observed that shRNA-mediated inhibition of XBP1 or ATF4 induces AML cell apoptosis and significantly extends disease latency in vivo tying the reduced survival mediated by JUN inhibition to the loss of pro-survival UPR signaling. These data uncover a previously unrecognized role of JUN as a regulator of the UPR as well as provide key new insights into the how ER stress responses contribute to AML and identify JUN and the UPR as promising therapeutic targets in this disease.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i’s), which are rarely curative. PARP1 inhibitors (PARP1i’s) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i’s. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i’s.

Myeloproliferative neoplasms (MPNs) often carry JAK2(V617F), MPL(W515L), or CALR(del52) mutations. Current treatment options for MPNs include cytoreduction by hydroxyurea and JAK1/2 inhibition by ruxolitinib, both of which are not curative. We show here that cell lines expressing JAK2(V617F), MPL(W515L), or CALR(del52) accumulated reactive oxygen species-induced DNA double-strand breaks (DSBs) and were modestly sensitive to poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673. At the same time, primary MPN cell samples from individual patients displayed a high degree of variability in sensitivity to these drugs. Ruxolitinib inhibited 2 major DSB repair mechanisms, BRCA-mediated homologous recombination and DNA-dependent protein kinase-mediated nonhomologous end-joining, and, when combined with olaparib, caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of patients. Moreover, the combination of BMN673, ruxolitinib, and hydroxyurea was highly effective in vivo against JAK2(V617F)+ murine MPN-like disease and also against JAK2(V617F)+, CALR(del52)+, and MPL(W515L)+ primary MPN xenografts. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized MPN cells to synthetic lethality triggered by PARP inhibitors.

Key Points PARP1 is required for the maintenance of MLL-AF9 leukemias. PARP1 inhibitors enhance the therapeutic effect of cytotoxic drugs against MLL-AF9 leukemias.

Clinical resistance to tyrosine kinase inhibitors (TKI) remains a significant problem in the therapy of patients with chronic myeloid leukemia (CML). Although BCR-ABL1-kinase domain mutations are the major cause of resistance to TKI, some patients manifest primary or develop secondary resistance to TKI without detectable BCR-ABL1 mutations. We aimed to assess the prevalence of additional gene mutations in a group of 50 patients with primary (n = 26) or secondary resistance (n = 24) to TKI, in most cases to imatinib (n = 49) and in one to dasatinib. We employed ~1000 genes custom target enrichment kit and next-generation sequencing on Illumina platform, as well as Sanger sequencing. In 21 patients, we were able to match and analyze paired samples collected at diagnosis (before treatment) and at the time of resistance and in five other cases we analyzed additional samples collected later during TKI-resistant chronic phase or in blast crisis. The most frequent genetic aberrations detected at the time of TKI-resistance were mutations in ASXL1 and BCR-ABL1 kinase domain coding sequence, present in 28% (14/50) and 26% (13/50) of patients, respectively. Both genes were mutated in 12% of patients (6/50), while 30% (15/50) had either ASXL1 or BCR-ABL1 mutations alone. Non-recurrent genetic aberrations in other genes were also noted in single patients (e.g. DNMT3A mutations). According to COSMIC database, all but three ASXL1 mutations detected in this study were previously described in hematologic malignancies and all (including the novel ones) are predicted to introduce stop codons or cause frameshifts in codon range of 512-943, thus truncating the protein before the C-terminal PHD domain. Among patients with available paired samples three had more than one ASXL1 mutation at various time points and in all of them we observed dynamics of specific ASXL1 mutations, related partially to the leukemic cell content in the sample but also providing evidence of clonal evolution (Table 1). In most of patients with concurrent ASXL1 and BCR-ABL1 mutations, we observed that both mutations were present with similar variant allele frequency (VAF), suggesting that ASXL1 mutations occur in Philadelphia-positive leukemic clones (Table 2). ASXL1 mutation frequency was significantly higher in TKI-resistant patients, than in our previously characterized group of CML patients without resistance, who achieved major molecular response (28% ; 14/50 vs 5.6% ; 2/36, respectively, p = 0.0105, Fisher exact test). All ASXL1-mutated patients with secondary (n = 1) or primary resistance (n = 5) for whom diagnostic sample was available, carried ASXL1 mutation also at the time of diagnosis. No significant difference in mutation frequency was found between patients with primary and secondary resistance (ASXL1 mutation, 26.9% vs 29.2% ; BCR-ABL1 mutation, 15.4% vs 37.5%). Our results provide evidence that preexisting ASXL1 mutations in BCR-ABL1-positive leukemic clone present at diagnosis may have impact on clinical response to imatinib and may be useful in assessing the risk of treatment failure. Disclosures Niesiobedzka-Krezel: Novartis: Honoraria. Seferynska:Novartis: Honoraria. Gora Tybor:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sacha:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background BRCA1/2 mutations are associated with several cancers. Our previous study indicate that microenvironmental stress conditions, induce BRCA1 protein deficiency not related to gene mutations, but resulting from attenuated translation. We propose that highly mortal pancreatic tumors might also belong to the BRCAness ones, even if not carrying BRCA1/2 mutations. In this study we developed and validated flow cytometry panel to estimate BRCA2 protein level, together with tumor and stem markers, to correlate with sensitivity to PARP1 inhibitors. Methods Antibody validation was performed in WT and mutant cells to setup the panel. Tumor cells were detected using CK19, CA19-9, CEACAM5 markers, together with CD44, CD133 and EpCAM for stem cell population, combined with viability dyes. For proof-of-concept experiments we used pancreatic adenocarcinoma (PDAC) cell lines: PANC-1 and BRCA2-defective CAPAN-1. Cells were treated with FDA approved Olaparib or BMN673, followed by apoptosis and cell cycle analysis. Finally, the primary cells from resected pancreas samples of PDAC patients were analysed in context of BRCA2 protein, tumor and stem cell markers expression. Results We found that selected BRCA-deficient cells show increased, dose-dependent, sensitivity to PARP1 inhibitors, compared to WT cells. This was manifested by significant G1 decrease, cell cycle arrest in G2/M and increased cell death induced by both drugs. Analysis of patient samples showed correlation between stem cell markers and BRCA2-deficiency. The validation process of antibody specificity, as well as the established protocol of cell isolation from pancreatic tumor samples will be also presented. Conclusions The developed flow cytometry panel can be applied to select BRCAness PDAC cells which has increased sensitivity to PARP1 inhibitors. Moreover, the selection of BRCA-deficient cohort based on flow cytometry test should be further investigated and considered as the diagnostic method for personalized therapies caused by PARP1 inhibitors. Legal entity responsible for the study The authors. Funding Foundation for Polish Science TEAM TECH Core Facility Plus/2017-2/2 (POIR.04.04.00-00-23C2/17-00) (KP). Disclosure All authors have declared no conflicts of interest.

Homologous recombination repair (HRR) protects cells from the lethal effect of spontaneous and therapy-induced DNA double-stand breaks. HRR usually depends on BRCA1/2-RAD51, and RAD52-RAD51 serves as back-up. To target HRR in tumor cells, a phenomenon called "synthetic lethality" was applied, which relies on the addiction of cancer cells to a single DNA repair pathway, whereas normal cells operate 2 or more mechanisms. Using mutagenesis and a peptide aptamer approach, we pinpointed phenylalanine 79 in RAD52 DNA binding domain I (RAD52-phenylalanine 79 [F79]) as a valid target to induce synthetic lethality in BRCA1- and/or BRCA2-deficient leukemias and carcinomas without affecting normal cells and tissues. Targeting RAD52-F79 disrupts the RAD52-DNA interaction, resulting in the accumulation of toxic DNA double-stand breaks in malignant cells, but not in normal counterparts. In addition, abrogation of RAD52-DNA interaction enhanced the antileukemia effect of already-approved drugs. BRCA-deficient status predisposing to RAD52-dependent synthetic lethality could be predicted by genetic abnormalities such as oncogenes BCR-ABL1 and PML-RAR, mutations in BRCA1 and/or BRCA2 genes, and gene expression profiles identifying leukemias displaying low levels of BRCA1 and/or BRCA2. We believe this work may initiate a personalized therapeutic approach in numerous patients with tumors displaying encoded and functional BRCA deficiency.

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). Unfortunately, 25% of TKI-naive patients and 50-90% of patients developing TKI-resistance carry CML clones expressing TKI-resistant BCR-ABL1 kinase mutants. We reported that CML-CP leukemia stem and progenitor cell populations accumulate high amounts of reactive oxygen species, which may result in accumulation of uracil derivatives in genomic DNA. Unfaithful and/or inefficient repair of these lesions generates TKI-resistant point mutations in BCR-ABL1 kinase. Using an array of specific substrates and inhibitors/blocking antibodies we found that uracil DNA glycosylase UNG2 were inhibited in BCR-ABL1-transformed cell lines and CD34(+) CML cells. The inhibitory effect was not accompanied by downregulation of nuclear expression and/or chromatin association of UNG2. The effect was BCR-ABL1 kinase-specific because several other fusion tyrosine kinases did not reduce UNG2 activity. Using UNG2-specific inhibitor UGI, we found that reduction of UNG2 activity increased the number of uracil derivatives in genomic DNA detected by modified comet assay and facilitated accumulation of ouabain-resistant point mutations in reporter gene Na(+)/K(+)ATPase. In conclusion, we postulate that BCR-ABL1 kinase-mediated inhibition of UNG2 contributes to accumulation of point mutations responsible for TKI resistance causing the disease relapse, and perhaps also other point mutations facilitating malignant progression of CML.

Fanconi D2 (FANCD2) is monoubiquitinated on K561 (FANCD2-Ub) in response to DNA double-strand breaks (DSBs) to stimulate repair of these potentially lethal DNA lesions. FANCD2-Ub was upregulated in CD34+ chronic myeloid leukemia (CML) cells and in BCR-ABL1 kinase-positive cell lines in response to elevated levels of reactive oxygen species (ROS) and DNA cross-linking agent mitomycin C. Downregulation of FANCD2 and inhibition of FANCD2-Ub reduced the clonogenic potential of CD34+ CML cells and delayed BCR-ABL1 leukemogenesis in mice. Retarded proliferation of BCR-ABL1 positive FANCD2-/- leukemia cells could be rescued by FANCD2 expression. BCR-ABL1 positive FANCD2-/- cells accumulated more ROS-induced DSBs in comparison with BCR-ABL1 positive FANCD2+/+ cells. Antioxidants diminished the number of DSBs and enhanced proliferation of BCR-ABL1 positive FANCD2-/- cells. Expression of wild-type FANCD2 and FANCD2(S222A) phosphorylation-defective mutant (deficient in stimulation of intra-S phase checkpoint, but proficient in DSB repair), but not FANCD2(K561R) monoubiquitination-defective mutant (proficient in stimulation of intra-S phase checkpoint, but deficient in DSB repair) reduced the number of DSBs and facilitated proliferation of BCR-ABL1 positive FANCD2-/- cells. We hypothesize that FANCD2-Ub has an important role in BCR-ABL1 leukemogenesis because of its ability to facilitate the repair of numerous ROS-induced DSBs.

BCR-ABL1 kinase-induced chronic myeloid leukemia in chronic phase (CML-CP) usually responds to treatment with ABL tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, and nilotinib. In most patients TKIs reduce the leukemia cell load substantially, but some leukemia cells, for example leukemia stem cells (LSCs), are intrinsically refractory to TKIs. In addition, some patients who respond initially may later become resistant to TKIs due to accumulation of point mutations in BCR-ABL1 kinase. LSCs or their progeny, leukemia progenitor cells (LPCs), at some stage may acquire additional genetic changes that cause the leukemia to transform further to a more advanced blast phase (CML-BP), which responds poorly to treatment and is usually fatal. We postulate that LSCs and/or LPCs refractory or resistant to TKIs may be 'ticking time-bombs' accumulating additional genetic aberrations and eventually 'exploding' to generate additional TKI-resistant clones and CML-BP clones with complex karyotypes.

Bone marrow microenvironment (BMM) also defined as a stem cell niche provides a major obstacle for current anti-leukemia treatment modalities including tyrosine kinase inhibitors (TKIs) such as imatinib. We recently reported that chronic myeloid leukemia (CML) stem cells are sensitive to PARP inhibitor (PARPi)-triggered synthetic lethality in conditions mimicking peripheral blood (normoxia, no stromal cells). We reported that PARPi olaparib and talazoparib eliminate imatinib-refractory CML cells in the conditions mimicking BMM (hypoxia, stromal cells present). The effect, however, is not as robust as that observed in normoxia implicating BMM-specific protective impact. Therefore, more efficient targeting of PARP is required to enhance synthetic lethal effect against CML in BMM. Most PARPi's have been designed to compete with NAD for a binding site on the PARP1 molecule. This strategy resulted in the discovery of nucleotide-like PARPi's that not only target PARP, but, unfortunately, other enzymatic pathways involving NAD and other nucleotides as co-factors. Using such inhibitors affects multiple NAD/nucleotide-dependent enzymatic pathways, which results in secondary toxic effects proceeding from the inactivation of other pathways, while the efficiency against the PARP pathway is diminished. Thus, the challenge is to design inhibitors based on other activities of PARP1. We reported that DSBs-dependent interaction of PARP1 with histone 4 (H4) resulted in activation of PARP1 enzymatic activity and stimulation of Alt-NHEJ. Using high-throughput screen we identified non-NAD-like inhibitors, which interfered with H4-mediated activation of PARP1 and were effective against several types of tumors including BRCA1-deficient CML cells. Here we show that combination of two structurally different PARPi's, NAD-like olaparib or talazoparib and non-NAD-like 5F02 resulted in more abundant elimination of CML cells and reduced toxicity to normal counterparts. Finally, the combination exerted synergistic effect in humanized immunodeficient mice bearing primary CML xenografts. Altogether, non-NAD-like PARP1i synergistically enhanced synthetic lethal effect of NAD-like PARPi in tumor cells without increasing the cytotoxic effect in normal cells. While synthetic lethality mediated by NAD-like PARPi is usually associated with enhanced accumulation of potentially lethal DSBs, non-NAD-like PARP1i 5F02 did not induce DSBs when used as a single agent and also in combination with olaparib. Thus, the mechanistic aspect of the synergistic effect of NAD-like and non-NAD-like PARPis needs to be elucidated. Nevertheless, our data suggest that combining NAD-like and allosteric non-NAD-like PARPi's may represent a viable therapeutic strategy for enhancing CML response to PARP inhibition and reducing the resistance that inevitably results from treatment with NAD-like PARPi's alone. Disclosures Valent: Novartis: Honoraria; Incyte: Honoraria; Pfizer: Honoraria.

Nbs1, a member of the Mre11-RAD50-Nbs1 complex, is phosphorylated by ATM, the product of the ataxia-telangiectasia mutated gene and a member of the phosphatidylinositol 3-kinase–related family of serine-threonine kinases, in response to DNA double-strand breaks (DSBs) to regulate DNA damage checkpoints. Here we show that BCR/ABL stimulated Nbs1 expression by induction of c-Myc–dependent transactivation and protection from caspase-dependent degradation. BCR/ABL-related fusion tyrosine kinases (FTKs) such as TEL/JAK2, TEL/PDGFβR, TEL/ABL, TEL/TRKC, BCR/FGFR1, and NPM/ALK as well as interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) also stimulated Nbs1 expression. Enhanced ATM kinase–dependent phosphorylation of Nbs1 on serine 343 (S343) in response to genotoxic treatment was detected in leukemia cells expressing BCR/ABL and other FTKs in comparison to normal counterparts stimulated with IL-3, GM-CSF, and SCF. Expression of Nbs1-S343A mutant disrupted the intra–S-phase checkpoint, decreased homologous recombinational repair (HRR) activity, down-regulated XIAP expression, and sensitized BCR/ABL-positive cells to cytotoxic drugs. Interestingly, inhibition of Nbs1 phosphorylation by S343A mutant enhanced the antileukemia effect of the combination of imatinib and genotoxic agent.

Genes encoding c-ABL kinase and BCR protein are targeted by yet-unknown mechanisms causing DNA double-strand breaks resulting in the generation of a chimeric gene encoding BCR/ABL fusion tyrosine kinase. BCR/ABL kinase displays transforming activity because of its constitutive kinase activity causing deregulated proliferation, apoptosis, differentiation, and adhesion. Moreover, BCR/ABL kinase is able to facilitate DNA repair, prolong activation of G2/M and S cell cycle checkpoints, and elevate expression of the antiapoptotic protein Bcl-X(L), making malignant cells less responsive to antitumor treatment. BCR/ABL may also stimulate generation of reactive oxygen species and enhance spontaneous DNA damage in tumor cells. Unfortunately, BCR/ABL kinase compromises the fidelity of DNA repair mechanisms, thus contributing to the accumulation of additional genetic abnormalities that lead to resistance to inhibitors such as imatinib mesylate and to malignant progression of the disease. Therefore, chronic myelogenous leukemia cells display mutator phenotype.

Mutations in the BCR/ABL kinase domain play a major role in resistance to imatinib mesylate (IM). We report here that BCR/ABL kinase stimulates reactive oxygen species (ROS), which causes oxidative DNA damage, resulting in mutations in the kinase domain. The majority of mutations involved A/T→G/C and G/C→A/T transitions, a phenotype detected previously in patients, which encoded clinically relevant amino acid substitutions, causing IM resistance. This effect was reduced in cells expressing BCR/ABL(Y177F) mutant, which does not elevate ROS. Inhibition of ROS in leukemia cells by the antioxidants pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), and vitamin E (VE) decreased the mutagenesis rate and frequency of IM resistance. Simultaneous administration of IM and an antioxidant exerted better antimutagenic effect than an antioxidant alone. Therefore, inhibition of ROS should diminish mutagenesis and enhance the effectiveness of IM. (Blood. 2006;108:319-327)

Objective BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias. We show that a signaling protein, phosphatidylinositol-3 kinase (PI-3k), is essential for growth of CML cells, but not of normal hematopoietic cells, and that p85α subunit of PI-3k co-immunoprecipitates with BCR/ABL. Therefore, we made an attempt to better characterize p85α–BCR/ABL interactions. Materials and Methods The mutants of p85α-SH3 domain were generated by in vitro site-directed mutagenesis system. Protein lysates were obtained from p210BCR/ABL-transformed murine 32Dcl3 myeloid cells, and in vitro transcription/translation was used to produce BCR/ABL protein. Pull-down and Western analyses were performed to detect the interaction between BCR/ABL and p85α-SH3. BCR/ABL-transformed 32Dcl3 cells were infected with internal ribosome entry site-green fluorescent protein retroviral construct encoding p85α-SH3 mutants to assess their biological effects. Results We show here that the SH3 domain of p85α (p85α-SH3) pulls down the p210BCR/ABL kinase from hematopoietic cell lysates. The interaction between p85α-SH3 and BCR/ABL may be intermediated by proteins such as c-Cbl, Shc, Grb2, and/or Gab2. Mutations in the p85α-SH3 region responsible for proline-rich motif binding either abrogate or enhance these interactions. These mutants exert a modest inhibitory effect on growth factor-independent proliferation of BCR/ABL-positive 32Dcl3 cells. Conclusions Based on this information we speculate on the capability of p85α-SH3 to interact with the protein network of BCR/ABL oncoprotein.

Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGF beta R and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic myelogenous leukemias and non-Hodgkin's lymphoma. Murine hematopoietic growth factor dependent BaF3 cells and cells transformed by FTK (BaF3-FTK) were used to investigate the role of FTKs in response to DNA damage. FTK-transformed cells displayed resistance to genotoxic treatment including gamma-radiation and cytostatic agents such as idarubicin and MNNG. More FTK-transformed cells survived genotoxic treatment and were able to proliferate in comparison to parental non-transformed cells. Similar or higher levels of DNA damage was detected in gamma-irradiated in BaF3-FTK cells in comparison to BaF3 parental cells. Idarubicin induced different amounts of DNA damage in various BaF3-FTK cells. All BaF3-FTK cells treated with MNNG displayed significantly more DNA damage in comparison to BaF3 cells. Despite the extent of genotoxic effect BaF3-FTK cells were often able to repair damaged DNA more efficiently that the non-transformed counterparts. Inhibition of BCR/ABL kinase activity by STI571 (Gleevec, inatinib mesylate) abrogated the resistance to genotoxic treatment and inhibited DNA repair mechanisms. We hypothesize that facilitation of the DNA repair in FTK-positive cells may contribute to their resistance to genotoxic treatment.

BCR/ABL oncogenic tyrosine kinase activates STAT5, which plays an important role in leukemogenesis. The downstream effectors of the BCR/ABL→STAT5 pathway remain poorly defined. We show here that expression of the antiapoptotic protein A1, a member of the Bcl-2 family, and the serine/threonine kinase pim-1 are enhanced by BCR/ABL. This up-regulation requires activation of STAT5 by the signaling from SH3+SH2 domains of BCR/ABL. Enhanced expression of A1 and pim-1 played a key role in the BCR/ABL-mediated cell protection from apoptosis. In addition, pim-1 promoted proliferation of the BCR/ABL-transformed cells. Both A1 and pim-1 were required to induce interleukin 3–independent cell growth, inhibit activation of caspase 3, and stimulate cell cycle progression. Moreover, simultaneous up-regulation of both A1 and pim-1 was essential for in vitro transformation and in vivo leukemogenesis mediated by BCR/ABL. These data indicate that induction of A1 and pim-1 expression may play a critical role in the BCR/ABL-dependent transformation.

AKT-induced reactive oxygen species generate imatinib-resistant clones emerging from chronic myeloid leukemia progenitor cells

Burkitt lymphoma/leukemia (BL) is a highly aggressive mature B-cell neoplasm characterized by chromosomal rearrangements of the c-myc oncogene resulting in the overexpression of MYC transcription factor. The most common translocation is the t(8;14)(q24;q32) (85% of all cases), which involves MYC and IGH loci to generate IGH/MYC. Deregulation of MYC, a potent proto-oncogene and transcriptional regulator contributes to lymphomagenesis through alterations in cell cycle regulation, and cell differentiation, apoptosis, adhesion, and metabolism. BL treatment consists of high-intensity chemotherapy protocols that include cyclophosphamide, cytarabine (AraC) and doxorubicin. Current therapies have achieved a very favorable outcome resulting in complete remission in 75% to 90% of BL patients and a survival rate of 70% to 80%. However the current treatment for BL is suboptimal in elderly patients or patients with advanced-stage diseased, in the setting of HIV infections, as well as in the setting of relapsed disease. Therefore new therapeutic strategies are necessary to improve the outcomes in BL diseases in the poor prognosis patients. It has been reported that overexpression of MYC caused accumulation of potentially lethal DNA double-strand breaks (DSBs), which can modulate the response of tumor cells to genotoxic treatment. Therefore, we examined the consequences of DSBs accumulation in IGH/MYC-positive BL cells. Here we show that untreated and cytarabine (AraC)-treated IGH/MYC-positive BL cells accumulate high number of potentially lethal DNA double-strand breaks (DSBs) and display downregulation of BRCA2 tumor suppressor protein, which is a key element of homologous recombination - mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro. Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary BL xenografts. In conclusion, we postulate that BRCA2 deficiency may predispose BL cells to synthetic lethality triggered by PARP1 inhibitor, such as recently FDA approved olaparib. Moreover, PARP1 inhibitor may be useful for the treatment of other malignancies associated with deregulation of MYC, including diffuse large B-cell lymphoma (DLBCL) and ALK-positive LBCL. Disclosures Wasik: Gilead Sciences: Equity Ownership; Seattle Genetics: Honoraria; Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: NPM-ALK as an omncogene; University of Pennsylvania: Patents & Royalties: CAR T-cells; Gilead Sciences: Research Funding; Pharmacyclics: Research Funding.

The ability of BCR-ABL oncoproteins to induce leukemic transformation of hematopoietic cells depends on their tyrosine kinase activity, which is essential for recruitment and activation of multiple pathways that transduce oncogenic signals. Although it is unknown yet whether activation of PI 3-kinase is required for transformation, the colony-forming ability of Philadelphia cells is dependent on PI 3-kinase activity, as indicated by the results of studies using a number of strategies to interfere with the synthesis and/or the function of the regulatory and catalytic subunits of this kinase. In particular, wortmannin, a specific PI 3-kinase inhibitor, preferentially affected colony formation of Philadelphia cells over that of normal marrow hematopoietic progenitors. The mechanism(s) of such effects are unknown, but PI 3-kinase inhibitors may represent a novel class of therapeutic agents for the ex vivo and/or in vivo treatment of Philadelphia leukemias.

The BCR/ABL oncogenic tyrosine kinase is responsible for initiating and maintaining the leukemic phenotype of Philadelphia chromosome (Ph1)- positive cells. Phosphatidylinositol-3 (PI-3) kinase is known to interact with and be activated by receptor and nonreceptor tyrosine kinases. We investigated whether PI-3 kinase associates with and/or is regulated by BCR/ABL, whether this interaction is functionally significant for Ph1 cell proliferation, and, if so, whether inhibition of PI-3 kinase activity can be exploited to eliminate Ph1-positive cells from bone marrow. We show that the p85 alpha subunit of PI-3 kinase associates with BCR/ABL and that transient expression of BCR/ABL in fibroblasts and down-regulation of BCR/ABL expression using antisense oligodeoxynucleotides (ODNs) in Ph1 cells activates and inhibits, respectively, PI-3 kinase enzymatic activity. The use of specific ODNs or antisense constructs to downregulate p85 alpha expression showed a requirement for p85 alpha subunit in the proliferation of BCR/ABL-dependent cell lines and chronic myelogenous leukemia (CML) primary cells. Similarly, wortmannin, a specific inhibitor of the enzymatic activity of the p110 subunit of PI-3 kinase, inhibited growth of these cells. The growth of normal bone marrow and erythromyeloid, but not megakaryocyte, progenitors was inhibited by p85 alpha antisense [S]ODNs, but wortmannin, at the concentrations tested, did not affect normal hematopoiesis. The proliferation of two BCR/ABL- and growth factor-independent cell lines was not affected by downregulation of the expression of the p85 alpha subunit or inhibition of p110 enzymatic activity, confirming the specificity of the observed effects on Ph1 cells. Thus, PI-3 kinase is one of the downstream effectors of BCR/ABL tyrosine kinase in CML cells. Moreover, reverse transcriptase-polymerase chain reaction performed on single colonies to detect BCR-ABL transcripts showed that wortmannin was able to eliminate selectively CML-blast crisis cells from a mixture of normal bone marrow and Ph1 cells.

Following the 53rd annual meeting of the American Society of Hematology (ASH) in San Diego in December 2011, a group of clinical and laboratory investigators convened for the 6th Post-ASH International Workshop on Chronic Myeloid Leukemia (CML) and Myeloproliferative Neoplasms (MPN). The Workshop took place on 13-14 December at the Estancia, La Jolla, California, USA. This report summarizes the most recent advances in the biology and therapy of CML that were presented at the ASH meeting and discussed at the Workshop. Preclinical studies focused on the CML stem cell and its niche, and on early results of deep sequencing of CML genomes. Clinical advances include updates on second- and third-generation tyrosine kinase inhibitors (TKIs), molecular monitoring, TKI discontinuation studies and new therapeutic agents. A report summarizing the pertinent advances in MPN has been published separately. © 2013 Informa UK, Ltd.

The mitochondrial respiratory chain (MRC) consists of protein complexes I, II, III, IV and V that support oxidative phosphorylation (OXPHOS), which depends on electron transport to generate adenosine triphosphate (ATP). Electron "leakage" from the MRC generates reactive oxygen species (ROS). Chronic myeloid leukemia in chronic phase (CML-CP) stem cells (LSCs) produce high levels of mitochondrial ROS, causing oxidative DNA damage, resulting in genomic instability, generating imatinib-resistant BCR-ABL1 kinase mutants and additional chromosomal aberrations. Using global mRNA microarray analysis combined with Ingenuity Pathway Analysis we found that LSCs display enhanced expression of genes encoding MRC complexes I, II, IV and V. However, expression of genes encoding complex III was deregulated. Treatment with imatinib did not correct the aberrant levels of MRC genes. Therefore we postulate that abnormal expression of MRC genes may facilitate electron "leakage" to promote the production of ROS and accumulation of genomic instability in LSCs in imatinib-naive and imatinib-treated patients.

Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII–generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor–resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)–positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

The BCR-ABL1 oncogenic tyrosine kinase can transform pluripotent hematopoietic stem cells and initiate chronic myeloid leukemia in chronic phase (CML-CP), a myeloproliferative disorder characterized by excessive accumulation of mature myeloid cells. Patients in CML-CP usually respond to treatment with ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, though some patients who respond initially may become resistant later. CML-CP leukemia stem cells (LSCs) are intrinsically insensitive to TKIs and thus survive in the long term. These LSCs or their progeny may at some stage acquire additional genetic changes that cause the leukemia to transform further, from CML-CP to a more advanced phase, which has been subclassified as either accelerated phase (CML-AP) or blastic phase (CML-BP). CML-BP is characterized by a major clonal expansion of immature progenitors, which have either myeloid or lymphoid features. CML-BP responds poorly to treatment and is usually fatal. This review discusses the role of genomic instability leading to blastic transformation of CML and proposes some novel therapeutic approaches.

Altered DNA repair mechanisms are responsible for survival of leukemia stem cells (LSCs) and/or leukemia progenitor cells (LPCs) accumulating numerous lethal DNA double-strand breaks (DSBs). DSBs resulting from stalled/broken replication forks in proliferating cells are primarily repaired by RAD51-mediated homologous recombination repair (HR), which depends on BRCA1-PALB2-BRCA2-RAD51 paralogs (BRCA pathway), while RAD52 pathway serves as redundant back-up mechanism. Enhanced self-renewal of LSCs and high proliferation rate of LPCs commit them to HR. It has been reported that inhibition of RAD52 either by the knockout, specific shRNA, or a small peptide aptamer induced synthetic lethality in BRCA pathway-deficient tumor cell lines and primary leukemia cells. Yet pharmacological inhibition of RAD52, which binds single-stranded DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we applied high-throughput screening and structure-based selection followed by biochemical assays and computer modeling to identify three leading compounds: (1) 20264, (2) RU-0084339, and (3) D-I03. Compound 20264 appeared to interact with the hotspot in RAD52 DNA binding domain 1 to interfere with ssDNA binding. RU-0084339 is a major allosteric inhibitor of RAD52 ssDNA binding domain which disassembles undecamer ring structure of RAD52. D-I03 abrogated RAD52-mediated ssDNA annealing and ssDNA pairing. RAD52 small molecule inhibitor (RAD52smi) reduced recruitment of RAD52 to DNA damage-induced nuclear foci and suppressed RAD52-mediated DNA double-strand break (DSB) repair activity in cells with negligible effects on other DSB repair pathways. Importantly, RAD52smi selectively eliminated cancer cell lines carrying BRCA1/2 inactivating mutations. Since inactivating mutations in BRCA pathway are rare in leukemias, individual BRCA pathway-deficient leukemias were identified by Gene Expression and Mutation Analysis (GEMA). Gene Expression approach applied microarrays, qRT-PCR, and/or flow cytometry to identify individual leukemias displaying downregulation of at least one gene in BRCA pathway. On the other hand, Gene Mutation strategy detected individual leukemias expressing an oncogene causing downregulation of BRCA pathway gene(s) (e.g., BCR-ABL1, MLL-AF9, AML1-ETO - mediated downregulation of BRCA1 and/or BRCA2) and harboring inactivating mutations in BRCA pathway (e.g., BRCA2 = FANCD1, and other FA genes). BRCA-deficient cells from individual patients indentified by GEMA were selectively sensitive to RAD52smi alone or in combination with already approved cytotoxic drugs. RAD52 is a promising new target because it is "druggable" by small molecule inhibitors. Moreover, inhibition of RAD52 by genetic knockout and small peptide aptamer did not exert any major negative effects in normal cells and tissues. Altogether, this work provided foundation for precision medicine guided synthetic lethality in BRCA-deficient leukemias exerted by small molecule inhibitors targeting novel mechanism - RAD52 dependent DSB repair. Disclosures No relevant conflicts of interest to declare.

Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" a majority of CML patients due to the presence of TKI-refractory quiescent leukemia stem cells (LSCs) and/or TKI-resistant proliferating LSCs. Moreover, a sub-population of patients does not respond favorably to TKI therapy and/or accumulates TKI-resistant BCR-ABL1 mutants and additional chromosomal aberrations (high risk/poor responders = HR/PR). This process may lead to the disease relapse and/or malignant progression to fatal chronic myeloid leukemia in accelerated phase or blast phase (CML-AP/BP). Therefore new treatment modalities are necessary to improve therapeutic outcome of CML HR/PRs. We found that CML LSCs and LPCs, including quiescent LSCs, from HR/PRs accumulate the highest levels of reactive oxygen species (ROS)-induced DNA double strand breaks (DSBs) in comparison to "good responders" and normal counterparts (Bolton-Gillespie et al., Blood, 2013). DSBs are the most lethal DNA lesions, but CML cells can tolerate them because two major repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), are hyper-activated (Slupianek et al., Mol. Cell, 2001; Oncogene, 2005; DNA Repair, 2006; Cancer Res., 2011; Blood, 2011; Nowicki et al., Blood, 2005). It appears that CML cells, and especially these from HR/PRs are "addicted" to DSB repair pathways to survive pro-apoptotic challenge from lethal DSBs. There are critical differences between DSB repair in normal and CML cells due to downregulation of BRCA1 in HR pathway and DNA-Pkcs in NHEJ pathway (Slupianek et al., Cancer Res., 2011; Podszywalow-Bartnicka et al., Cell Cycle, 2014). Therefore, proliferating LSCs/LPCs employ RAD52-dependent alternative HR, in contrast to BRCA1-mediated HRR in normal counterparts. Quiescent LSCs use PARP1-mediated alternative NHEJ instead of DNA-PKcs -dependent NHEJ, which is predominant in normal quiescent HSCs. We hypothesized that simultaneous targeting of PARP1 and RAD52 will trigger "dual synthetic lethality" eradicating quiescent LSCs and proliferating LSCs/LPCs. To test this hypothesis we generated Parp1-/-Rad52-/- double knockout mice, which did not display any detectable problems in hematopoietic system and other organs. Leukemogenesis in SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice was much prolonged in comparison to SCLtTA/p210BCR-ABL1/Parp1-/-, SCLtTA/p210BCR-ABL1/Rad52-/- and SCLtTA/p210BCR-ABL1 animals; 33% of SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice did not develop leukemia. Moreover, BCR-ABL1 transfected Parp1-/-Rad52-/- bone marrow cells formed the lowest number of colonies when compared to BCR-ABL1 transfected Parp1-/-, Rad52-/-, and wild-type cells. Next, we tested anti-leukemia effect of a combination of PARP1 inhibitor (FDA approved Lynparza) and RAD52 small molecule inhibitor (RAD52smi) identified by high-throughput screen. Simultaneous administration of RAD52 and PARP1 inhibitors completely eradicated clonogenic activity of TKI-treated CML-CP HR/PRs and CML-AP LSCs/LPCs and almost exhausted quiescent LSCs without affecting normal cells. These inhibitors were also very effective against Ph+ ALL cells. In conclusion "dual synthetic lethality" triggered by simultaneous targeting of PARP1 and RAD52 represents a novel strategy which may eradicate quiescent LSCs and proliferating LSCs/LPCs not only from CML but also from BCR-ABL1 -positive ALL. Disclosures Valent: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria.

BCR-ABL1 kinase-positive leukemia cells accumulate high numbers of DNA double-strand breaks (DSBs) induced by the reactive oxygen species (ROS) or cytotoxic agents. To repair these lesions and prevent apoptosis BCR-ABL1 kinase stimulates the efficiency of DSB repair in leukemia cells. Histone acetylation-dependent chromatin re-modeling plays a crucial role in this process. Here we report that leukemia cells expressing BCR-ABL1 kinase displayed an enhanced histone acetylase activity (HAT) and reduced histone deacetylase activity (HDAC), which was associated with abundant expression of acetylated histones 3 and 4 (Ac-H3 and Ac-H4, respectively). Moreover, Ac-H3 and Ac-H4 readily co-localized with the spontaneous and mitomycin C-induced DSBs in BCR-ABL1-positive leukemia cells suggesting that histone acetylation and chromatin re-modeling is important for efficient repair of numerous DSBs.

NIH Public Access Author Manuscript Am J Hematol. Author manuscript; available in PMC 2012 November 01. Published in final edited form as: Am J Hematol. 2011 September ; 86(9): 811–819. doi:10.1002/ajh.22097. $watermark-text Chronic Myeloid Leukemia 2011: Successes, challenges, and strategies – Proceedings of the 5 th Annual BCR-ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms workshop Tariq I Mughal 1 , Jerald P Radich 2 , Richard A. Van Etten 3 , Alfonso Quintas-Cardama 4 , Tomasz Skorski 5 , Farhad Ravandi 6 , Daniel J. DeAngelo 7 , Carlo Gambacorti-Passerini 8 , Giovanni Martinelli 9 , and Ayalew Tefferi 10 1 University of Colorado School of Medicine, Denver, USA 2 Fred Hutchinson Cancer Center, Seattle, USA 3 Tufts Medical Center, Boston, USA 4 MD Anderson Cancer Center, Houston, USA $watermark-text 5 Temple 6 MD University, Philadelphia, USA Anderson Cancer Center, Houston, USA 7 Dana Farber Cancer Center at Harvard Medical School, Boston, USA 8 University 9 Insitute 10 Mayo of Milan, Monza, Italy Le A. Seragnoli, Bologna, Italy Clinic, Rochester, MN, USA. Abstract $watermark-text This report is based on the presentations and discussions at the 5 th annual BCR-ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms (MPN) workshop, which took place immediately following the 52 nd American Society of Hematology (ASH) meeting in Orlando, Florida on December 7 th -8 th , 2011. Relevant data which was presented at the ASH meeting as well as all other recent publications were presented and discussed at the workshop. This report covers front-line therapies of BCR-ABL1-positive leukemias, in addition to addressing some topical biological, pre-clinical and clinical issues, such as new insights into genomic instability and resistance to tyrosine kinase inhibitors (TKIs), risk stratification and optimizing molecular monitoring. A report pertaining to the new therapies and other pertinent preclinical and clinical issues in the BCR-ABL1 negative MPNs is published separately. Introduction For patients with BCR-ABL1-positive leukemias, which comprise of all the Philadelphia (Ph) chromosome-positive and some Ph-negative leukemias, the introduction of the original tyrosine kinase inhibitor (TKI), imatinib mesylate (IM) into the clinics in 1998, resulted in being a classic therapeutic landmark (1,2). After 12 months of therapy with IM, 69% of Correspondence to: Tariq Mughal MD FRCP FACP 23655 Currant Drive, Golden, Colorado 80401, USA Tel +1 303 526 8586 tmughal911@hotmail.com.

BCR/ABL fusion tyrosine kinase transforms hematopoietic stem cells causing chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). BCR/ABL regulates numerous proteins involved in apoptosis, proliferation and cell - cell or cell - extracellular matrix interactions. However, BCR/ABL also enhances DNA damage caused by endogenous reactive oxygen species and exogenous genotoxic treatment. In addition, BCR/ABL modulates the response to DNA damage to promote genomic instability. This function leads to resistance to ABL kinase small molecular inhibitors (SMIs) imatinib (IM), dasatinib and nilotinib, and contributes to malignant progression of the disease. The former phenomenon is often caused by mutations in BCR/ABL kinase whereas the latter is associated with accumulation of additional genetic aberrations including chromosomal translocations, deletions, additional chromosomes, gene amplifications, and point mutations. Possible benefits of anti-mutagenic therapy used in pursuing the cure of BCR/ABL-positive leukemias are discussed.

BCR-ABL1 fusion tyrosine kinase transforms hematopoietic stem cells (HSCs) and cause chronic myeloid leukemia in chronic phase (CML-CP), which is a stem cell (leukemia stem cell=LSC) -derived but a progenitor (leukemia progenitor cell=LPC)-driven disease. Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, nilotinib and ponatinib revolutionized the treatment of CML. We showed that reactive oxygen species (ROS) may increase the levels oxidative DNA damage in TKI-naïve and TKI-treated Lin-CD34+CD38- LSCs, including quiescent LSCs, and in Lin-CD34+CD38+ LPCs. Since DNA repair mechanisms display reduced fidelity/activity in CML cells, overproduction of ROS may promote genomic instability (accumulation of point mutations and chromosomal aberrations) resulting in resistance to TKIs and malignant progression of the disease to accelerated (CML-AP) or blast (CML-BP) phase. BCR-ABL1 kinase stimulates numerous signaling pathways to induce and maintain transformation of hematopoietic cells. We and others found that phosphatidylinositol-3 kinase (PI3k) plays an essential role not only in growth factor independent proliferation and protection from apoptosis of CML cells, but also in overproduction of mitochondrial ROS. Here we investigated the role of PI3k downstream effectors, AKT serine/threonine kinase and Rac GTPase in genomic instability in TKI-naïve and TKI-treated LSCs and LPCs. Using CML-CP primary cells and an inducible model of murine CML-CP –like disease we determined that Rac2 – PAK serine/threonine pathway alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III thereby generating high levels of ROS in TKI-naïve and TKI-treated LSCs and LPCs. In addition, AKT appeared to play an essential role in generation of ROS-induced oxidative DNA damage in TKI-naïve and TKI-treated LPCs, but not in LSCs. Inhibition of AKT did not affect the activity of Rac2 and inhibition of Rac2 did not affect AKT, clearly indicating that their activation status does not depend on each other. Targeting Rac2 and AKT either by mutations/deletions/shRNAs or small molecule inhibitors/peptides reduced genomic instability resulting in diminished frequency of TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations. Altogether TKI-naive and TKI-treated LSCs (including quiescent LSCs) display only PI3k-Rac2-PAK pathway, whereas LPCs contain PI3k-Rac2-PAK and PI3k-AKT pathways responsibble for ROS-induced oxidative DNA damage and genomic instability. Our findings may have more broad application because other oncogenic tyrosine kinases expressed in hetatopoietic malignancies induce similar mechanisms of genomic instability. Disclosures No relevant conflicts of interest to declare.

The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, & that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes. Disclosures: No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs), and especially quiescent LSCs, have a dual role as tumor initiating and therapy-refractory cells. Therefore, even if anti-tumor treatment clears a disease burden consisting mostly of proliferating leukemia progenitor cells (LPCs), it usually fails to eradicate therapy-refractory LSCs and also therapy-resistant LPCs. LSCs, including quiescent LSCs and LPCs accumulate high numbers of spontaneous and drug-induced DNA lesions, including highly lethal DNA double-strand breaks (DSBs). Thus, LSCs and/or LPCs may be “addicted” to particular DNA repair mechanisms and targeting these pathways could sensitize LSCs to the lethal effect of unrepaired DSBs. DSBs are usually repaired by two major mechanisms, homologous recombination repair (HRR) and non-homologous end-joining (NHEJ). While NHEJ plays a major role in quiescent cells, HRR works predominantly on broken replication forks in proliferating cells. Normal quiescent cells employ DNA-PK (PRKDC, XRCC5, XRCC6, LIG4) –mediated NHEJ to repair DSBs whereas PARP1-dependent NHEJ serves as back-up. Normal proliferating cells use PARP1-mediated base excision repair (BER) to prevent DSBs at collapsed replication forks, whereas BRCA (BRCA1, BRCA2, PALB2, RAD51 paralogs, RAD51)-mediated HRR serves as back-up to repair stalled/broken forks. The existence of these pathways creates the opportunity to apply “synthetic lethality” triggered by PARP1 inhibitors (PARP1i) in DNA-PK –deficient therapy-refractory quiescent LSCs and BRCA-deficient therapy-resistant proliferating LSCs/LPCs. This hypothesis is supported by PARP1i-mediated synthetic lethality in BRCA1-/- proliferating and XRCC6-/-quiescent cells. Since inactivating mutations in BRCA and DNA-PK pathways are rather rare in leukemias we hypothesize that Gene Expression and Mutation Analysis (GEMA) can identify individual patients with leukemias displaying “spontaneous” and oncogene-induced downregulation of DSB repair genes which will be sensitive to “synthetic lethality” triggered by PARP1i +/- already approved drugs. GENE EXPRESSION ANALYSIS: Global mRNA microarrays and qRT-PCR followed by immunofluorescent protein staining identified primary AMLs and ALLs from individual patients displaying BRCA- and/or DNA-PK –deficient phenotype (downregulation of at least one member of BRCA and/or DNA-PK pathway). Quiescent DNA-PK –deficient LSCs and proliferating BRCA-deficient LSCs/LPCs, but not DNA-PK and BRCA –proficient counterparts were sensitive to olaparib and BMN673 +/- already approved drugs. GENE MUTATION ANALYSIS: BCR-ABL1 downregulates BRCA1 and DNA-PKcs proteins, FLT3(ITD) inhibits BRCA1 protein, and MLL-AF9 and AML1-ETO reduce BRCA2 and XRCC6 proteins, whereas HOXA9+MEIS1 do not affect the expression of DSB repair proteins. These effects were associated with reduced colony formation by BCR-ABL1, MLL-AF9 and AML1-ETO –transformed, but not HOXA9+MEIS1-treasformed Parp1-/-murine bone marrow cells. PARP1i olaparib and BMN673 +/- approved drugs induced DSBs resulting in abrogation of cell proliferation and/or apoptosis in DNA-PK –deficient quiescent LSCs and/or BRCA-deficient proliferating LSCs/LPCs from CML-CP/AP, Ph+ B-ALL, AML and ALL patients. Genetic (Parp1-/-) and pharmacological (olaparib, BMN673) inhibition of PARP1 prolonged survival of mice with CML-CP –like inducible disease and reduced engraftment/prolonged survival of NSG immunodeficient mice with DNA-PK and/or BRCA-deficient primary leukemia (CML-CP, AML and ALL) xenografts in combination with approved drugs. In conclusion, targeting PARP1 resulted in synthetic lethality in quiescent LSCs and/or proliferating LSCs/LPCs from individual patients identified by GEMA, which display specific defects in DSB repair mechanisms. Disclosures Minden: Celgene: Honoraria.

Anti-oxidant vitamin E prevents accumulation of imatinib-resistant BCR-ABL1 kinase mutations in CML-CP xenografts in NSG mice

The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph)-positive leukemia cells. We find that BCR/ABL-induced reactive oxygen species (ROSs) cause chronic oxidative DNA damage resulting in double-strand breaks (DSBs) in S and G2/M cell cycle phases. These lesions are repaired by BCR/ABL-stimulated homologous recombination repair (HRR) and nonhomologous end-joining (NHEJ) mechanisms. A high mutation rate is detected in HRR products in BCR/ABL-positive cells, but not in the normal counterparts. In addition, large deletions are found in NHEJ products exclusively in BCR/ABL cells. We propose that the following series of events may contribute to genomic instability of Ph-positive leukemias: BCR/ABL → ROSs → oxidative DNA damage → DSBs in proliferating cells → unfaithful HRR and NHEJ repair.