Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.

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 IC50 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.

R-Loop Accumulation in Spliceosome Mutant Leukemias Confers Sensitivity to PARP1 Inhibition by Triggering Transcription-Replication Conflicts.

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.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological 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 induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal 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 to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

Epigenetic balance ensures mechanistic control of MLL amplification and rearrangement.

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.

Class I HDAC Inhibition Leads to a Downregulation of FANCD2 and RAD51, and the Eradication of Glioblastoma Cells.

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.

Histone Deacetylases (HDAC) Inhibitor-Valproic Acid Sensitizes Human Melanoma Cells to Dacarbazine and PARP Inhibitor.

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.

Simultaneous Targeting of DNA Polymerase Theta and PARP1 or RAD52 Triggers Dual Synthetic Lethality in Homologous Recombination-Deficient Leukemia Cells.

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.

TIAR and FMRP shape pro-survival nascent proteome of leukemia cells in the bone marrow microenvironment.

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.

Haploinsufficiency of ZNF251 causes DNA-PKcs-dependent resistance to PARP inhibitors in BRCA1-mutated cancer cells.

Poly (ADP-ribose) polymerase (PARP) inhibitors represent a promising new class of agents that have demonstrated efficacy in treating various cancers, particularly those that carry BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPis) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, resistance to PARPis is common and can occur through multiple mechanisms, including the restoration of HR and/or the stabilization of replication forks. To gain a better understanding of the mechanisms underlying PARPi resistance, we conducted an unbiased CRISPR-pooled genome-wide library screen to identify new genes whose deficiency confers resistance to the PARPi olaparib. Our study revealed that ZNF251, a transcription factor, is a novel gene whose haploinsufficiency confers PARPi resistance in multiple breast and ovarian cancer lines harboring BRCA1 mutations. Mechanistically, we discovered that ZNF251 haploinsufficiency leads to constitutive stimulation of DNA-PKcs-dependent non-homologous end joining (NHEJ) repair of DSBs and DNA-PKcs-mediated fork protection in BRCA1-mutated cancer cells (BRCA1mut + ZNF251KD). Moreover, we demonstrated that DNA-PKcs inhibitors can restore PARPi sensitivity in BRCA1mut + ZNF251KD cells ex vivo and in vivo. Our findings provide important insights into the mechanisms underlying PARPi resistance and highlight the unexpected role of DNA-PKcs in this phenomenon.

ABL1 kinase as a tumor suppressor in AML1-ETO and NUP98-PMX1 leukemias.

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.

DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.

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.

Pre-Existing and Acquired Resistance to PARP Inhibitor-Induced Synthetic Lethality.

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.

Synthetic Lethality Targeting Polθ.

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.

Perspective: Pivotal translational hematology and therapeutic insights in chronic myeloid hematopoietic stem cell malignancies.

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.

4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells.

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.

Polθ Inhibition: An Anticancer Therapy for HR-Deficient Tumours.

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.

TET2 and DNMT3A Mutations Exert Divergent Effects on DNA Repair and Sensitivity of Leukemia Cells to PARP Inhibitors.

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 FLT3ITD, BCR-ABL1, JAK2V617F , and MPLW515L , or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52 . 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.

ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.

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.