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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.

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.

Supplementary Figure S1. BL #1 cells display low HR activity. Supplementary Figure S2. BL #1 induced BL-like leukemia in NSG mice Supplementary Figure S3. IGH/MYC-positive cells are hypersensitive to RAD52 inhibitor. Supplementary Figure S4. IGH/MYC does not downregulate mRNA encoding BRCA2. Supplementary Figure S5. c-MYC regulates the expression of BRCA2 protein. Supplementary Figure S6. miR-1245 is upregulated in IGH/MYC-positive cells.

Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC–positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC–positive cells was associated with diminished HR activity and 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 Burkitt lymphoma xenografts. In conclusion, IGH/MYC–positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC–induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpg.Mol Cancer Res; 15(8); 967–72. ©2017 AACR.

Supplemental Figure Legends

Supplemental Methods

Supplemental Figure 7. (A-D) OCI-AML3 cells were transduced with lentiviruses expressing CTRL shRNA or SOD2 shRNA and analyzed for: (A) Western blot analysis 4 days after transduction with the indicated antibodies. (B) MitoSOX and Annexin V staining by flow cytometry, 4 days after transduction. The bar graph shows the MitoSOX median fluorescence intensity (MFI) of live (Annexin V-) cells and data is expressed as mean {plus minus} SD of three independent experiments. (C) Cell growth every 2 days by MTS analysis. Data represents the mean {plus minus} SD of three independent replicates. (D) Annexin V staining 5 days after transduction. Data represents the mean {plus minus} SD of three independent replicates. (E) Bar graph representing the number of colonies formed by mouse MLL-AF9 leukemia cells transduced with CTRL shRNA, Sod2 shRNA_1 and Sod2 shRNA_2 in methylcellulose culture. Data is expressed as mean {plus minus} SD of three independent replicates. (*p{less than or equal to}0.05, **p{less than or equal to}0.01; ****p

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.

Supplemental Figure 6. (A & B) OCI-AML3 cells were transduced with CTRL shRNA, PKCε shRNA_1 or PKCε shRNA_2 and 24 hours later cells from each condition were treated with 100µM NAC (A) or 50µM glutathione (B), every 12 hours until the end of the experiment. Four days after treatment, cells from each condition were assessed for Annexin V staining by flow cytometry (****p

Supplementary Methods and Results

Supplemental Table 1. Clinical characteristics and mutation status of primary AML samples.

Purpose: The intracellular redox environment of acute myeloid leukemia (AML) cells is often highly oxidized compared to healthy hematopoietic progenitors and this is purported to contribute to disease pathogenesis. However, the redox regulators that allow AML cell survival in this oxidized environment remain largely unknown.Experimental Design: Utilizing several chemical and genetically-encoded redox sensing probes across multiple human and mouse models of AML, we evaluated the role of the serine/threonine kinase PKC-epsilon (PKCϵ) in intracellular redox biology, cell survival and disease progression.Results: We show that RNA interference-mediated inhibition of PKCϵ significantly reduces patient-derived AML cell survival as well as disease onset in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. We also show that PKCϵ inhibition induces multiple reactive oxygen species (ROS) and that neutralization of mitochondrial ROS with chemical antioxidants or co-expression of the mitochondrial ROS-buffering enzymes SOD2 and CAT, mitigates the anti-leukemia effects of PKCϵ inhibition. Moreover, direct inhibition of SOD2 increases mitochondrial ROS and significantly impedes AML progression in vivo. Furthermore, we report that PKCϵ over-expression protects AML cells from otherwise-lethal doses of mitochondrial ROS-inducing agents. Proteomic analysis reveals that PKCϵ may control mitochondrial ROS by controlling the expression of regulatory proteins of redox homeostasis, electron transport chain flux, as well as outer mitochondrial membrane potential and transport.Conclusions: This study uncovers a previously unrecognized role for PKCϵ in supporting AML cell survival and disease progression by regulating mitochondrial ROS biology and positions mitochondrial redox regulators as potential therapeutic targets in AML. Clin Cancer Res; 24(3); 608–18. ©2017 AACR.

Supplementary Figure 1 from BCR/ABL and Other Kinases from Chronic Myeloproliferative Disorders Stimulate Single-Strand Annealing, an Unfaithful DNA Double-Strand Break Repair

Supplementary Figures 1-4 from BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

Supplementary Tables 1-2, Figures 1-2 from Imatinib Sensitivity in BCR-ABL1–Positive Chronic Myeloid Leukemia Cells Is Regulated by the Remaining Normal ABL1 Allele

Supplementary Figure 2 from BCR/ABL Inhibits Mismatch Repair to Protect from Apoptosis and Induce Point Mutations

Supplementary Methods from BCR/ABL Stimulates WRN to Promote Survival and Genomic Instability

Supplementary Figure 3 from BCR/ABL Inhibits Mismatch Repair to Protect from Apoptosis and Induce Point Mutations

Supplementary Figure 1 from BCR/ABL Inhibits Mismatch Repair to Protect from Apoptosis and Induce Point Mutations

Supplementary Methods from Imatinib Sensitivity in BCR-ABL1–Positive Chronic Myeloid Leukemia Cells Is Regulated by the Remaining Normal ABL1 Allele

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.

Poly (ADP-ribose) polymerase (PARP) inhibitors represent a promising new class of agents that have demonstrated efficacy in treating various cancers, particularly those that carryBRCA1/2mutations. The cancer associatedBRCA1/2mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPis) have been applied to trigger synthetic lethality inBRCA1/2-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 thatZNF251haploinsufficiency leads to constitutive stimulation of DNA-PKcs-dependent non-homologous end joining (NHEJ) repair of DSBs and DNA-PKcs-mediated fork protection inBRCA1-mutated cancer cells (BRCA1mut+ZNF251KD). Moreover, we demonstrated that DNA-PKcs inhibitors can restore PARPi sensitivity in BRCA1mut+ZNF251KD cellsex vivoandin vivo. Our findings provide important insights into the mechanisms underlying PARPi resistance and highlight the unexpected role of DNA-PKcs in this phenomenon.Significance statementOur study identified a novel geneZNF251which haploinsufficiency causes the resistance to PARP inhibitors in breast cancer cells and pinpointed a novel mechanism of the resistance.

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.

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.

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.

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.

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.

The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase. Cytoplasmic ABL1 stimulates, whereas nuclear ABL1 inhibits cell growth. Analysis of the CGAP Mitelman database detected loss of ABL1 allele in hematologic malignancies. Among the top genetic aberrations detected in acute myeloid leukemias were t(8;21) generating fusion protein AML1-ETO and NUP98 translocations, e.g., t(1;11) encoding NUP98-PMX1.Expression of AML1-ETO and NUP98-PMX1 in Lin-cKit+ stem cell enriched bone marrow cells from Abl1-/-;Vav-Cre and Abl1+/+;Vav-Cre mice revealed that oncogene-expressing Abl1-/- cells gained tremendous proliferation advantage. Conversely, overexpression of ABL1 and DPH (ABL1 kinase agonist)-mediated activation of ABL1 kinase resulted in reduced proliferation of AML1-ETO and NUP98-PMX1 cells. Since the role of ABL1 in DNA damage response (DDR) is well established, we applied the inhibitors of ATM (ATMi), ATR (ATRi) and DNA-PKcs (DNA-PKi) to identify potential therapeutic vulnerabilities. AML1-ETO;Abl1-/- cells were more sensitive to all three inhibitors when compared to AML1-ETO;Abl1+/+ cells. Remarkably, NUP98-PMX1;Abl1-/- cells were exceptionally sensitive to ATRi, but not to ATMi and DNA-PKi when compared to NUP98-PMX1;Abl+/+ cells. ABL1 did not affect the sensitivity of transformed cells to DNA damaging agent doxorubicin. To pinpoint mechanisms collaborating with Abl1 deletion in transformation of hematopoietic cells, Abl1 was knockout in 32Dcl3 cells by CRISPR/Cas9 to obtain 32Dcl3-Abl1ko cells. 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells were challenged to induce transformation by starving them from growth factors (IL3). Only 32Dcl3-Abl1ko cell populations generated growth factor-independent cells. This indicates that the absence of Abl1 may facilitate malignant transformation of hematopoietic cells. RNA-seq was performed to compare gene expression in 32Dcl3-Abl1wt and 32Dcl3-Abl1ko cells in the presence or absence of IL-3. Pathway analysis of FPKM reads (top 30 significant canonical pathways) and Venn diagram analysis followed by KEGG pathway analysis (291 genes uniquely expressed in growth factor-independent 32Dcl3-Abl1ko cells) implicated PI3K-AKT pathway signaling including mTOR and sirtuins (SITR) in 32Dcl3-Abl1ko cells. PI3K inhibitor buparlisib, mTOR inhibitor rapamycin and SIRT2 inhibitor thiomyristoyl exerted selective activity against Lin-cKit+ AML1-ETO;Abl1-/- and/or NUP98-PMX1;Abl1-/- cells when compared to Abl1+/+ counterparts. Moreover, inhibition of ABL1 kinase by imatinib in Lin-cKit+ AML1-ETO;Abl1+/+ and/or NUP98-PMX1;Abl+/+ cells induced the sensitivity to PI3K-ATK pathway inhibitors. In conclusion, we showed that normal ABL1 kinase plays a tumor suppressor role and is a potential therapeutic target in hematological malignancies induced by AML1-ETO and NUP98-PMX1. The latter statement is being currently tested in animal models. Citation Format: Konstantin Golovine, Zhaorui Lian, Kumaraswamy Naidu Chitrala, Gleb Ablakov, Margaret Nieborowska-Skorska, Jian Huang, Tomasz Skorski. ABL1 kinase regulates leukemia phenotype and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 823.

BRCA1 jest jednym z głównych regulatorów stabilności genomowej w komórce. Odpowiada ze kontrolę segregacji chromosomów oraz komórek potomnych po podziale oraz reguluje naprawę dwuniciowych pęknęć DNA poprzez rekombinację homologiczną (HR). Zmiany genetyczne na poziomie genu BRCA1, takie jak mutacje i zmiany epigenetyczne są markerem predyspozycji w kierunku zachorowania na nowotwór piersi, jajnika i prostaty, istotnie zwiększając ryzyko wystąpienia nowotworu. Zmiany takie nie są obserwowane w białaczkach. Co ważne, deficyty BRCA1 są czynnikiem wskazującym na wrażliwość pacjentów na personalizowaną terapię inhibitorami PARP1, opartą o zjawisko tzw. syntetycznej letalności. W niniejszej pracy przedstawiamy nasze badania prowadzące do odkrycia nowego mechanizmu prowadzącego do deficytów BRCA1 w białaczkach, który nie jest związany z mutacjami lub innymi zmianami na poziomie genu, lecz z zahamowaną translacją i deficytem białka BRCA1. Zaburzenia te są efektem aktywacji komórkowej odpowiedzi na stres i kontrolowane przez białka wiążące RNA. Co więcej, wykazaliśmy, że pewne terapie jak i zmiany genetyczne w białaczkach także mogą wywołać deficyty BRCA1. Nasze badania dowiodły, że inhibitory PARP1 powinny być rozważane jako skuteczne terapeutyki w przypadku białaczek z niedoborem BRCA1, prowadząc do skutecznej eliminacji komórek nowotworu, w tym także komórek macierzystych i progenitorowych. W końcu, w wyniku naszych badań, zaproponowaliśmy strategię selekcji pacjentów z białaczkami, którzy będą wrażliwi na terapię inhibitorami PARP1.

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.

textabstractThe original version of this Article contained errors in Figure 6. In panel o, the labels incorrectly stated ‘Poleθ’ and “Poleθ + DNA” and should be labelled “Polθ” and “Polθ + DNA”. In the result section, in the sub-section entitled “Polθ Promotes MMEJ of Long ssDNA”, the sentence “Importantly, the ability of Polθ- pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates it performs interstrand pairing without Polθ-hel”. should read as follow: “Importantly, the ability of Polθ-pol to perform MMEJ on short (≤12 nt) ssDNA (Fig. 1p, left; Supplementary Fig. 3D and 3E), and short (≤15 nt) overhangs, demonstrates that it performs interstrand pairing without Polθ-hel”. In the sub-section entitled “Preventing Intrastrand Pairing Stimulates MMEJ by Polθ-Pol”, the sentence “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along long the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. should read as follows: “We predicted that preventing base-pairing opportunities between 3' terminal bases and bases upstream along the 5' region of long ssDNA substrates would suppress intrastrand pairing and enable interstrand pairing by Polθ-pol (Fig. 3c)”. In the method section, in the “Proteins” sub-section the sentence “Polθ-pol, Polθ-hel and RPA were purified as described”. should read as follows: “Polθ-pol and Polθ-hel were purified as described”. These corrections have now been included in the HTML and pdf of the article. Additionally, a technical problem during the publication process resulted in loss of image quality in Figs. 1, 3 and 4. This has now been corrected in both the PDF and HTML versions of the Article.

Summary: 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.