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Leukemia, although most likely starts as a monoclonal genetic/epigenetic anomaly, is a polyclonal disease at manifestation. This polyclonal nature results from ongoing evolutionary changes in the genome/epigenome of leukemia cells to promote their survival and proliferation advantages. We discuss here how genetic and/or epigenetic aberrations alter intracellular microenvironment in individual leukemia clones and how extracellular microenvironment selects the best fitted clones. This dynamic polyclonal composition of leukemia makes designing an effective therapy a challenging task especially because individual leukemia clones often display substantial differences in response to treatment. Here, we discuss novel therapeutic approach employing single cell multiomics to identify and eradicate all individual clones in a patient., (© 2024. The Author(s).)

Myeloid malignancies carrying somatic DNMT3A mutations (DNMT3Amut) are usually resistant to standard therapy. DNMT3Amut leukemia cells accumulate toxic DNA double strand breaks (DSBs) and collapsed replication forks, rendering them dependent on DNA damage response (DDR). DNA polymerase theta (Polθ), a key element in Polθ-mediated DNA end-joining (TMEJ), is essential for survival and proliferation of DNMT3Amut leukemia cells. Polθ is overexpressed in DNMT3Amut leukemia cells due to abrogation of PARP1 PARylation-dependent UBE2O E3 ligase-mediated ubiquitination and proteasomal degradation of Polθ. In addition, PARP1-mediated recruitment of the SMARCAD1-MSH2/MSH3 repressive complex to DSBs was diminished in DNMT3Amut leukemia cells which facilitated loading of Polθ on DNA damage and promoting TMEJ and replication fork restart. Polθ inhibitors enhanced the anti-leukemic effects of mainstream drugs such as FLT3 kinase inhibitor quizartinib, cytarabine and etoposide in vitro and in mice with FLT3(ITD);DNMT3Amut leukemia. Altogether, Polθ is an attractive target in DNMT3Amut hematological malignancies.

DNA repair proteins became the popular targets in research on cancer treatment. In our studies we hypothesized that inhibition of DNA polymerase theta (Polθ) and its combination with Poly (ADP-ribose) polymerase 1 (PARP1) or RAD52 inhibition and the alkylating drug temozolomide (TMZ) has an anticancer effect on glioblastoma cells (GBM21), whereas it has a low impact on normal human astrocytes (NHA). The effect of the compounds was assessed by analysis of cell viability, apoptosis, proliferation, DNA damage and cell cycle distribution, as well as gene expression. The main results show that Polθ inhibition causes a significant decrease in glioblastoma cell viability. It induces apoptosis, which is accompanied by a reduction in cell proliferation and DNA damage. Moreover, the effect was stronger when dual inhibition of Polθ with PARP1 or RAD52 was applied, and it is further enhanced by addition of TMZ. The impact on normal cells is much lower, especially when considering cell viability and DNA damage. In conclusion, we would like to highlight that Polθ inhibition used in combination with PARP1 or RAD52 inhibition has great potential to kill glioblastoma cells, and shows a synthetic lethal effect, while sparing normal astrocytes.

DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC 50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers., (© 2024. The Author(s).)

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ., (© 2024. The Author(s).)

Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL., (©2023 American Association for Cancer Research.)

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC 50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi., (© 2024. The Author(s).)

RNA splicing factor (SF) gene mutations are commonly observed in patients with myeloid malignancies. Here we showed that SRSF2- and U2AF1-mutant leukemias are preferentially sensitive to PARP inhibitors (PARPi), despite being proficient in homologous recombination repair. Instead, SF-mutant leukemias exhibited R-loop accumulation that elicited an R-loop-associated PARP1 response, rendering cells dependent on PARP1 activity for survival. Consequently, PARPi induced DNA damage and cell death in SF-mutant leukemias in an R-loop-dependent manner. PARPi further increased aberrant R-loop levels, causing higher transcription-replication collisions and triggering ATR activation in SF-mutant leukemias. Ultimately, PARPi-induced DNA damage and cell death in SF-mutant leukemias could be enhanced by ATR inhibition. Finally, the level of PARP1 activity at R-loops correlated with PARPi sensitivity, suggesting that R-loop-associated PARP1 activity could be predictive of PARPi sensitivity in patients harboring SF gene mutations. This study highlights the potential of targeting different R-loop response pathways caused by spliceosome gene mutations as a therapeutic strategy for treating cancer., Significance: Spliceosome-mutant leukemias accumulate R-loops and require PARP1 to resolve transcription-replication conflicts and genomic instability, providing rationale to repurpose FDA-approved PARP inhibitors for patients carrying spliceosome gene mutations., (©2023 American Association for Cancer Research.)

MLL/KMT2A amplifications and translocations are prevalent in infant, adult, and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here, we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the crosstalk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, in turn promoting amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications., Competing Interests: Declaration of interests J.R.W. has served or is serving as a consultant or advisor for Qsonica, Salarius Pharmaceuticals, Daiichi Sankyo, Inc., Vyne Therapeutics, and Lily Asia Ventures. J.R.W. also receives funding for research from Salarius Pharmaceuticals and Oryzon Genomics. O.G. is a scientific cofounder and shareholder of EpiCypher, Inc., K36 Therapeutics, Inc., and Alternative Bio, Inc. J.J. received research funds from Celgene Corporation, Levo Therapeutics, Inc., Cullgen, Inc., and Cullinan Oncology, Inc. J.J. is a cofounder and equity shareholder in Cullgen, Inc., a scientific cofounder and scientific advisory board member of Onsero Therapeutics, Inc., and a consultant for Cullgen, Inc., EpiCypher, Inc., and Accent Therapeutics, Inc. C.D. receives research funds from Janssen outside the submitted work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

DNA polymerase theta (Polθ, encoded by POLQ gene) plays an essential role in Polθ-mediated end-joining (TMEJ) of DNA double-strand breaks (DSB). Inhibition of Polθ is synthetic lethal in homologous recombination (HR)-deficient tumor cells. However, DSBs can be also repaired by PARP1 and RAD52-mediated mechanisms. Because leukemia cells accumulate spontaneous DSBs, we tested if simultaneous targeting of Polθ and PARP1 or RAD52 enhance the synthetic lethal effect in HR-deficient leukemia cells. Transformation potential of the oncogenes inducing BRCA1/2-deficiency (BCR-ABL1 and AML1-ETO) was severely limited in Polq-/-;Parp1-/- and Polq-/-;Rad52-/- cells when compared with single knockouts, which was associated with accumulation of DSBs. Small-molecule inhibitor of Polθ (Polθi) when combined with PARP or RAD52 inhibitors (PARPi, RAD52i) caused accumulation of DSBs and exerted increased effect against HR-deficient leukemia and myeloproliferative neoplasm cells., Implications: In conclusion, we show that PARPi or RAD52i might improve therapeutic effect of Polθi against HR-deficient leukemias., (©2023 American Association for Cancer Research.)

HDAC inhibitors (HDACi) hold great potential as anticancer therapies due to their ability to regulate the acetylation of both histone and non-histone proteins, which is frequently disrupted in cancer and contributes to the development and advancement of the disease. Additionally, HDACi have been shown to enhance the cytotoxic effects of DNA-damaging agents such as radiation and cisplatin. In this study, we found that histone deacetylase inhibits valproic acid (VPA), synergized with PARP1 inhibitor (PARPi), talazoparib (BMN-673), and alkylating agent, and temozolomide (TMZ) to induce DNA damage and reduce glioblastoma multiforme. At the molecular level, VPA leads to a downregulation of FANCD2 and RAD51, and the eradication of glioblastoma cells. The results of this study indicate that combining HDACi with PARPi could potentially enhance the treatment of glioblastoma, the most aggressive type of cancer that originates in the brain.

The inhibition of histone deacetylases (HDACs) holds promise as a potential anti-cancer therapy as histone and non-histone protein acetylation is frequently disrupted in cancer, leading to cancer initiation and progression. Additionally, the use of a histone deacetylase inhibitor (HDACi) such as the class I HDAC inhibitor-valproic acid (VPA) has been shown to enhance the effectiveness of DNA-damaging factors, such as cisplatin or radiation. In this study, we found that the use of VPA in combination with talazoparib (BMN-673-PARP1 inhibitor-PARPi) and/or Dacarbazine (DTIC-alkylating agent) resulted in an increased rate of DNA double strand breaks (DSBs) and reduced survival (while not affecting primary melanocytes) and the proliferation of melanoma cells. Furthermore, the pharmacological inhibition of class I HDACs sensitizes melanoma cells to apoptosis following exposure to DTIC and BMN-673. In addition, the inhibition of HDACs causes the sensitization of melanoma cells to DTIV and BMN-673 in melanoma xenografts in vivo. At the mRNA and protein level, the histone deacetylase inhibitor downregulated RAD51 and FANCD2. This study aims to demonstrate that combining an HDACi, alkylating agent and PARPi could potentially enhance the treatment of melanoma, which is commonly recognized as being among the most aggressive malignant tumors. The findings presented here point to a scenario in which HDACs, via enhancing the HR-dependent repair of DSBs created during the processing of DNA lesions, are essential nodes in the resistance of malignant melanoma cells to methylating agent-based therapies.

Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect., (© 2023 by The American Society of Hematology.)

Chronic myeloid leukemia (CML) cells circulate between blood and bone marrow niche, representing different microenvironments. We studied the role of the two RNA-binding proteins, T-cell-restricted intracellular antigen (TIAR), and the fragile X mental retardation protein (FMRP) in the regulation of protein translation in CML cells residing in settings mimicking peripheral blood microenvironment (PBM) and bone marrow microenvironment (BMM). The outcomes showed how conditions shaped the translation process through TIAR and FMRP activity, considering its relevance in therapy resistance. The QuaNCAT mass-spectrometric approach revealed that TIAR and FMRP have a discrete modulatory effect on protein synthesis and thus affect distinct aspects of leukemic cells functioning in the hypoxic niche. In the BMM setup, FMRP impacted metabolic adaptation of cells and TIAR substantially supported the resistance of CML cells to translation inhibition by homoharringtonine. Overall, our results demonstrated that targeting post-transcriptional control should be considered when designing anti-leukemia therapeutic solutions., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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., (© 2023. The Author(s).)

DNA polymerase theta (Polθ)-mediated end joining (TMEJ) is, along with homologous recombination (HR) and non-homologous end-joining (NHEJ), one of the most important mechanisms repairing potentially lethal DNA double-strand breaks (DSBs). Polθ is becoming a new target in cancer research because it demonstrates numerous synthetically lethal interactions with other DNA repair mechanisms, e.g., those involving PARP1, BRCA1/2, DNA-PK, ATR. Inhibition of Polθ could be achieved with different methods, such as RNA interference (RNAi), CRISPR/Cas9 technology, or using small molecule inhibitors. In the context of this topic, RNAi and CRISPR/Cas9 are still more often applied in the research itself rather than clinical usage, different than small molecule inhibitors. Several Polθ inhibitors have been already generated, and two of them, novobiocin (NVB) and ART812 derivative, are being tested in clinical trials against HR-deficient tumors. In this review, we describe the significance of Polθ and the Polθ-mediated TMEJ pathway. In addition, we summarize the current state of knowledge about Polθ inhibitors and emphasize the promising role of Polθ as a therapeutic target.

The advanced development of synthetic lethality has opened the doors for specific anti-cancer medications of personalized medicine and efficient therapies against cancers. One of the most popular approaches being investigated is targeting DNA repair pathways as the implementation of the PARP inhibitor (PARPi) into individual or combinational therapeutic schemes. Such treatment has been effectively employed against homologous recombination-defective solid tumors as well as hematopoietic malignancies. However, the resistance to PARPi has been observed in both preclinical research and clinical treatment. Therefore, elucidating the mechanisms responsible for the resistance to PARPi is pivotal for the further success of this intervention. Apart from mechanisms of acquired resistance, the bone marrow microenvironment provides a pre-existing mechanism to induce the inefficiency of PARPi in leukemic cells. Here, we describe the pre-existing and acquired mechanisms of the resistance to PARPi-induced synthetic lethality. We also discuss the potential rationales for developing effective therapies to prevent/repress the PARPi resistance in cancer cells.

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., (© 2022 John Wiley & Sons Ltd.)

Research studies regarding synthetic lethality (SL) in human cells are primarily motivated by the potential of this phenomenon to be an effective, but at the same time, safe to the patient's anti-cancer chemotherapy. Among the factors that are targets for the induction of the synthetic lethality effect, those involved in DNA repair seem to be the most relevant. Specifically, when mutation in one of the canonical DNA double-strand break (DSB) repair pathways occurs, which is a frequent event in cancer cells, the alternative pathways may be a promising target for the elimination of abnormal cells. Currently, inhibiting RAD52 and/or PARP1 in the tumor cells that are deficient in the canonical repair pathways has been the potential target for inducing the effect of synthetic lethality. Unfortunately, the development of resistance to commonly used PARP1 inhibitors (PARPi) represents the greatest obstacle to working out a successful treatment protocol. DNA polymerase theta (Polθ), encoded by the POLQ gene, plays a key role in an alternative DSB repair pathway-theta-mediated end joining (TMEJ). Thus, it is a promising target in the treatment of tumors harboring deficiencies in homologous recombination repair (HRR), where its inhibition can induce SL. In this review, the authors discuss the current state of knowledge on Polθ as a potential target for synthetic lethality-based anticancer therapies.

Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3 ITD , BCR-ABL1, JAK2 V617F , and MPL W515L , or with mutations affecting related signaling pathways such as NRAS G12D and CALR del52 . Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK-mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo , whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2-Wilms' tumor 1 (WT1)-binding ability was 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. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. SIGNIFICANCE: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase-positive malignant hematopoietic cells to PARP inhibitors., (©2021 American Association for Cancer Research.)

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism., Competing Interests: Declaration of interests S.P. is currently an employee of Merck Research Laboratories and R. Gabbasov is currently an employee of Carisma Therapeutics. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chronic myeloid leukemia (CML) develops due to the presence of the BCR-ABL1 protein, a target of tyrosine kinase inhibitors (TKIs), such as imatinib (IM), used in a CML therapy. CML eradication is a challenge due to developing resistance to TKIs. BCR-ABL1 induces endogenous oxidative stress leading to genomic instability and development of TKI resistance. Model CML cells susceptible or resistant to IM, as well as wild-type, non-cancer cells without the BCR-ABL1 protein were treated with IM, hydrogen peroxide (H 2 O 2 ) as a model trigger of external oxidative stress, or with IM+H 2 O 2 . Accumulation of reactive oxygen species (ROS), DNA damage, activity of selected antioxidant enzymes and glutathione (GSH), and mitochondrial potential (MMP) were assessed. We observed increase in ROS accumulation in BCR-ABL1 positive cells and distinct levels of ROS accumulation in IM-susceptible cells when compared to IM-resistant ones, as well as increased DNA damage caused by IM action in sensitive cells. Depletion of GSH levels and a decreased activity of glutathione peroxidase (GPx) in the presence of IM was higher in the cells susceptible to IM. IM-resistant cells showed an increase of catalase activity and a depletion of MMP. BCR-ABL1 kinase alters ROS metabolism, and IM resistance is accompanied by the changes in activity of GPx, catalase, and alterations in MMP.

DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The successful use of PARP1 inhibitors like olaparib (Loparza ® ) in the treatment of BRCA1/2- deficient breast cancer has provided clinical proof of concept for applying personalized medicine based on synthetic lethality to the treatment of cancer. Unfortunately, all marketed PARP1 inhibitors act by competing with the cofactor NAD + and resistance is already developing to this anti-cancer mechanism. Allosteric PARP1 inhibitors could provide a means of overcoming this resistance. A high throughput screen performed by Tulin et al. identified 5F02 as an allosteric PARP inhibitor that acts by preventing the enzymatic activation of PARP1 by histone H4. 5F02 demonstrated anti-cancer activity in several cancer cell lines and was more potent than olaparib and synergistic with olaparib in these assays. In the present study we explored the structure-activity relationship of 5F02 by preparing analogs that possessed structural variation in four regions of the chemical scaffold. Our efforts led to lead molecule 7 , which demonstrated potent anti-clonogenic activity against BRCA-deficient NALM6 leukemia cells in culture and a therapeutic index for the BRCA-deficient cells over their BRCA-proficient isogenic counterparts., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest.

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.

Alterations in DNA repair systems play a key role in the induction and progression of cancer. Tumor-specific defects in DNA repair mechanisms and activation of alternative repair routes create the opportunity to employ a phenomenon called "synthetic lethality" to eliminate cancer cells. Targeting the backup pathways may amplify endogenous and drug-induced DNA damage and lead to specific eradication of cancer cells. So far, the synthetic lethal interaction between BRCA1/2 and PARP1 has been successfully applied as an anticancer treatment. Although PARP1 constitutes a promising target in the treatment of tumors harboring deficiencies in BRCA1/2-mediated homologous recombination (HR), some tumor cells survive, resulting in disease relapse. It has been suggested that alternative RAD52-mediated HR can protect BRCA1/2-deficient cells from the accumulation of DNA damage and the synthetic lethal effect of PARPi. Thus, simultaneous inhibition of RAD52 and PARP1 might result in a robust dual synthetic lethality, effectively eradicating BRCA1/2-deficient tumor cells. In this review, we will discuss the role of RAD52 and its potential application in synthetic lethality-based anticancer therapies., Competing Interests: The authors declare no conflict of interest, financial, or otherwise.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely "cure" the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown 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. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1 -transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.

In our previous studies of the molecular mechanisms of poly(ADP-ribose) polymerase 1 (PARP-1)-mediated transcriptional regulation we identified a novel class of PARP-1 inhibitors targeting the histone-dependent route of PARP-1 activation. Because histone-dependent activation is unique to PARP-1, non-NAD-like PARP-1 inhibitors have the potential to bypass the off-target effects of classical NAD-dependent PARP-1 inhibitors, such as olaparib, veliparib, and rucaparib. Furthermore, our recently published studies demonstrate that, compared to NAD-like PARP-1 inhibitors that are used clinically, the non-NAD-like PARP-1 inhibitor 5F02 exhibited superior antitumor activity in cell and animal models of human prostate cancer (PC). In this study, we further evaluated the antitumor activity of 5F02 and several of its novel analogues against PC cells. In contrast to NAD-like PARP-1 inhibitors, non-NAD-like PARP-1 inhibitors demonstrated efficacy against androgen-dependent and -independent routes of androgen receptor signaling activation. Our experiments reveal that methylation of the quaternary ammonium salt and the presence of esters were critical for the antitumor activity of 5F02 against PC cells. In addition, we examined the role of a related regulatory protein of PARP-1, called Poly(ADP-ribose) glycohydrolase (PARG), in prostate carcinogenesis. Our study reveals that PARG expression is severely disrupted in PC cells, which is associated with decreased integrity and localization of Cajal bodies (CB). Overall, the results of our study strengthen the justification for using non-NAD-like PARP-1 inhibitors as a novel therapeutic strategy for the treatment of advanced prostate cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cancer is a heterogeneous disease with a high degree of diversity between and within tumors. Our limited knowledge of their biology results in ineffective treatment. However, personalized approach may represent a milestone in the field of anticancer therapy. It can increase specificity of treatment against tumor initiating cancer stem cells (CSCs) and cancer progenitor cells (CPCs) with minimal effect on normal cells and tissues. Cancerous cells carry multiple genetic and epigenetic aberrations which may disrupt pathways essential for cell survival. Discovery of synthetic lethality has led a new hope of creating effective and personalized antitumor treatment. Synthetic lethality occurs when simultaneous inactivation of two genes or their products causes cell death whereas individual inactivation of either gene is not lethal. The effectiveness of numerous anti-tumor therapies depends on induction of DNA damage therefore tumor cells expressing abnormalities in genes whose products are crucial for DNA repair pathways are promising targets for synthetic lethality. Here, we discuss mechanistic aspects of synthetic lethality in the context of deficiencies in DNA double strand break repair pathways. In addition, we review clinical trials utilizing synthetic lethality interactions and discuss the mechanisms of resistance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) - an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK-mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ., Competing Interests: CONFLICTS OF INTEREST There is no conflicts of interest.

BRCA1 (breast cancer 1 susceptibility protein) is one of main regulators of cellular genomic stability. It is responsible for proper segregation of chromatides to daughter cells during mitosis as well as DNA double strand breaks repair by homologous recombination (HR). Genetic alterations of BRCA1 gene are cancer predisposition markers. Mutations or epigenetic alterations have been noticed in breast, ovarian and prostate cancers, significantly increasing risk of cancer development. Such gene alterations are not connected with leukemias. Importantly, BRCA1 deficiency is a factor which makes patients susceptible for personalized therapy with PARP1 inhibitors, which is based on the phenomenon called synthetic lethality. In this review we present our discoveries of novel mechanism leading to BRCA1 deficiency in leukemia, which is not connected with BRCA1 gene mutations or epigenetic alterations, but with attenuated translation of BRCA1 protein linked to the cellular stress response and controlled by RNA binding proteins. Moreover, we found that some treatments or genetic alterations in leukemias might also result in BRCA1 deficits. Our studies provide evidence that PARP1 inhibitors should be considered as efficient treatment in BRCA1-deficient leukemias, leading to elimination of cancer cells, including stem and progenitor cells. Finally we propose a strategy to select leukemia patients which might be sensitive to therapy with PARP1 inhibitors.

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., (© 2018 by The American Society of Hematology.)