scholarly journals hCINAP regulates the DNA-damage response and mediates the resistance of acute myelocytic leukemia cells to therapy

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ruidan Xu ◽  
Shuyu Yu ◽  
Dan Zhu ◽  
Xinping Huang ◽  
Yuqi Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Leukemia Cells ◽  
Acute Myelocytic Leukemia ◽  
Myelocytic Leukemia ◽  
Damage Response

scholarly journals The SF3B1 K700E Mutation Induces R-Loop Accumulation and Associated DNA Damage

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4219-4219 ◽  
Author(s):  
Shalini Singh ◽  
Doaa Ahmed ◽  
Hamid Dolatshad ◽  
Dharamveer Tatwavedi ◽  
Ulrike Schulze ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
K562 Cells ◽  
Splicing Factor ◽  
Leukemia Cells ◽  
Cd34 Cells ◽  
Damage Response ◽  
Healthy Control ◽  
Atr Activation ◽  
Mutant Cells

The myelodysplastic syndromes (MDS) are common myeloid malignancies. Mutations in genes involved in pre-mRNA splicing (SF3B1, SRSF2, U2AF1 and ZRSR2) are the most common mutations found in MDS. There is evidence that some spliceosomal components play a role in the maintenance of genomic stability. Splicing is a transcription coupled process; splicing factor mutations affect transcription and may lead to the accumulation of R-loops (RNA-DNA hybrids with a displaced single stranded DNA). Mutations in the splicing factors SRSF2 and U2AF1 have been recently shown to increase R-loops formation in leukemia cell lines, resulting in increased DNA damage, replication stress and activation of the ATR-Chk1 pathway. SF3B1 is the most frequently mutated splicing factor gene in MDS, but a role for mutated SF3B1 in R-loop accumulation and DNA damage has not yet been reported in hematopoietic cells. We have investigated the effects of the common SF3B1 K700E mutation on R-loop formation and DNA damage response in MDS and leukemia cells. R-loop signals and the DNA damage response were measured by immunofluorescence staining using S9.6 and anti-γ-H2AX antibodies respectively. Firstly, we studied K562 (myeloid leukemia) cells with the SF3B1 K700E mutation and isogenic SF3B1 K700K wildtype (WT) K562 cells. K562 cells with SF3B1 mutation showed a significant increase in the number of S9.6 foci [Fold change (FC) 2.01, p<0.001] and in the number of γ-H2AX foci (FC 2.32, p<0.001), indicating increased R-loops and DNA damage, compared to SF3B1 WT K562 cells. Moreover, we observed increased Chk1 phosphorylation at Ser345, a hallmark of activation of the ATR pathway, in SF3B1 mutant K562 cells. Next, we analyzed induced pluripotent stem cells (iPSCs) that we generated from the bone marrow cells of one SF3B1 mutant MDS patient and of one healthy control. A significant increase in R-loops and DNA damage response was observed in an iPSC clone harboring SF3B1 mutation compared to another iPSC clone without SF3B1 mutation obtained from same MDS patient (S9.6 mean fluorescence intensity - FC 1.72, p<0.001; γ-H2AX foci - FC 1.34, p=0.052) and to iPSCs from the healthy control (S9.6 mean fluorescence intensity - FC 1.53, p<0.001; γ-H2AX foci - FC 1.61, p=0.006). In addition, bone marrow CD34+ cells from a SF3B1 mutant MDS patient showed increased R-loops (as measured by number of S9.6 foci) compared to CD34+ cells from a MDS patient without splicing factor mutations (FC 1.9) and from a healthy control (FC 2.6). To investigate whether the observed DNA damage and ATR activation in SF3B1 mutant K562 cells result from induced R-loops, we overexpressed RNASEH1 (encoding an enzyme that degrades the RNA in RNA:DNA hybrids) to resolve R-loops in these cells. RNASEH1 overexpression significantly reduced the number of S9.6 (FC 0.51, p<0.001) and γ-H2AX foci (FC 0.63, p=0.035) in SF3B1 mutant K562 cells compared to SF3B1 WT K562 cells. RNASEH1 overexpression also resulted in decreased Chk1 phosphorylation, indicating suppression of ATR pathway activation in SF3B1 mutant K562 cells. To determine the functional importance of ATR activation associated with SF3B1 mutation, we evaluated the sensitivity of SF3B1 mutant cells towards the ATR inhibitor VE-821. SF3B1 mutant K562 cells showed preferential sensitivity towards VE-821 compared to SF3B1 WT K562 cells. Chk1 is a critical substrate of ATR, and we next investigated the effects of Chk1 inhibition in SF3B1 mutant cells. Interestingly, SF3B1 mutant K562 cells demonstrated preferential sensitivity towards the Chk1 inhibitor UCN-1 (IC50 61.8 nM) compared to SF3B1 WT K562 cells (IC50 267 nM), suggesting that ATR activation is important for the survival of SF3B1 mutant cells. SF3B1 mutant K562 cells were preferentially sensitive to the splicing modulator Sudemycin D6 (IC50 53.2 nM) compared to SF3B1 WT K562 cells (IC50 130.7 nM). The effects of VE-821 and UCN-1 on SF3B1 mutant K562 cells were enhanced by Sudemycin D6 (Combination index <1), indicating synergy. In summary, our results show that the SF3B1 mutation leads to accumulation of R-loops and associated DNA damage resulting in activation of the ATR pathway in MDS and leukemia cells. Thus different mutated splicing factors have convergent effects on R-loop elevation leading to DNA damage. Moreover, our data suggest that Chk1 inhibition, alone or in combination with splicing modulators, may represent a novel therapeutic strategy to target SF3B1 mutant cells. Disclosures Schuh: Janssen: Speakers Bureau; Verastem: Speakers Bureau; Kite: Speakers Bureau; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy, Speakers Bureau; Genentech: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Speakers Bureau. Wiseman:Novartis, Celgene: Consultancy, Honoraria.

scholarly journals DNA damage response involves modulation of Ku70 and Rb functions by cyclin A1 in leukemia cells

2007 ◽  
Vol 121 (4) ◽  
pp. 706-713 ◽  
Author(s):  
Ping Ji ◽  
Nicole Bäumer ◽  
Taijun Yin ◽  
Sven Diederichs ◽  
Feng Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Leukemia Cells ◽  
Cyclin A1 ◽  
Damage Response

scholarly journals Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells

Oncotarget ◽  
2017 ◽  
Vol 8 (10) ◽  
pp. 16259-16274 ◽  
Author(s):  
Prexy Modi ◽  
Kumudha Balakrishnan ◽  
Qingshan Yang ◽  
William G. Wierda ◽  
Michael J. Keating ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Damage Response ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Damage Response

Targeting mTOR by a New Generation of Kinase Inhibitors Sensitizes Leukemia Cells for Chemotherapy Via Suppressing FANCD2 Expression

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1514-1514
Author(s):  
Jie Li ◽  
Fukun Guo ◽  
Jared Sipple ◽  
Sara Kozma ◽  
George Thomas ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Cancer Cell ◽  
Dna Damage Response ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Cancer Cell Proliferation ◽  
Damage Response

Abstract Abstract 1514 The mammalian target of rapamycin (mTOR) is a key regulator of nutrient metabolism, cell growth and proliferation. Inhibition of mTOR signaling by rapamycin or rapamycin in combination with antineoplastic agents has been shown to block cancer cell proliferation and cancer angiogenesis. Second-generation pharmacological mTOR inhibitors, which inhibit both mTORC1 and mTORC2 by directly targeting the ATP-binding site of mTOR, have recently shown improved activity in tumor suppression and are under clinical development for cancer therapy. Indeed, it is found that the mTOR kinase inhibitor PP242 inhibits cell proliferation more effectively than rapamycin in pre-clinical models, suggesting the additive contributions of mTORC1 and mTORC2 to cancer cell proliferation and survival. In the present study, we have explored the therapeutic value of PP242 in sensitizing tumor suppression by anti-cancer drugs. We found that the combination of PP242 with Cytarabine (AraC) or Etoposide induced significant higher apoptosis than single-agent treatment in several human lymphoma and leukemia cell lines including K562, Molt-Luc2, and K562-Luc2. Specifically, using Molt-Luc2 cells, the percentages of apoptosis for combined PP242-AraC and PP242-Etoposide treatments were 76.3±4.2% and 78.2+5.9%, respectively, in comparison with 52.7±6.3% and 38.2±4.5%, respectively, in AraC- and Etoposide-treated cells; the basal level of apoptosis in these leukemic cells was 5–8%. Further, PP242, but not Rapamycin, sensitized the leukemia and lymphoma cells to DNA damage induced by AraC or Etoposide, evidenced by a marked increase in g-H2AX foci (94±5% cells in PP242-AraC group vs 25±3% cells in AraC-alone group or 98±4% cells in PP242- Etoposide group vs 36±3% cells in Etoposide-alone group), as well as DNA-strand breaks (comet-tailed value of 25.4±1.2% in PP242-AraC group and 31.2±3.2% in PP242-Etoposide group compared to 9.8±1.2% in AraC-alone and 11.4±2.8% Etoposide-alone groups, respectively). This increased DNA damage response can be attributed to a suppression of the expression of FANCD2, a critical DNA damage repair component of the Fanconi pathway, by PP242, in both normal lymphoblasts and leukemic cells. Significantly, the effect of PP242 on Fanconi gene expression was FANCD2-specific as PP242 had no effect on the expression of other Fanconi proteins such as FANCA and FANCC, and forced expression of FANCD2 by a viral promoter completely abolished the sensitizing effect of PP242 on drug-induced leukemia cell death. We are currently using a mouse xenotransplant model to explore the in vivo effect of the combination of PP242 with AraC for human leukemia cells. Our findings suggest that the mTOR kinase inhibitor PP242 enhances antitumor activity of conventional chemo-drugs by suppressing FANCD2 and associated DNA damage response and consequently augmenting DNA damage leading to apoptosis. Therefore, PP242 combined with chemotherapy could represent a novel strategy for the treatment of hematopoietic malignancies. Disclosures: No relevant conflicts of interest to declare.

Enhancers of Polycomb EPC1 and EPC2 Prevent Accumulation of MYC to Sustain the Oncogenic Potential of MLL Leukemia Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 400-400
Author(s):  
Xu Huang ◽  
Tim Somervaille
Keyword(s):  
Stem Cells ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Dna Damage Response ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Oncogenic Potential ◽  
Damage Response ◽  
Stem And Progenitor Cells ◽  
Complex Components

Abstract Abstract 400 Dysregulation of the structure and function of chromatin is common in myeloid malignancy. We used a lentiviral screening strategy to target for knockdown ∼270 genes coding for chromatin regulatory proteins in human THP-1 AML cells. THP-1 cells harbor a t(9;11) translocation which is the cytogenetic hallmark of MLL-AF9, found in ∼5% of patients with AML. The strategy identified the EP400 chromatin complex components EPC1 and EPC2 as critical oncogenic co-factors in MLL leukaemia. Knockdown of both EPC1 and EPC2 in human MLL-mutated cell lines and of Epc1 and Epc2 in experimentally initiated murine MLL-AF9 AML cells led to loss of AML cell clonogenic potential, due to induction of apoptosis. The phenotype was rescued by forced expression of human EPC1 or EPC2 in Epc knockdown cells. Epc1 and Epc2 knockdown MLL-AF9 AML cells failed to initiate AML upon syngeneic transplantation into secondary murine recipients, while control cells initiated short latency leukemia. Likewise, xenogeneic transplantation of EPC1 or EPC2 knockdown primary human MLL-AF9 AML failed to engraft recipients, while control cells which engrafted every recipient to variable extent. In complete contrast, knockdown of EPC1 or EPC2 did not adversely impact on either the in vitro clonogenic or in vivo repopulating potential of primary murine KIT+ or human CD34+ normal haematopoietic stem and progenitor cells. These data demonstrate a remarkable selective dependency of MLL-mutated leukemia stem cells, but not normal hematopoietic stem and progenitor cells, on Epc1 and Epc2. EPC1 and EPC2 are part of an EP400 centred complex with a role in the DNA damage response. The complex is also known to associate with MYC. Consistent with a role for EPC containing complexes in the DNA damage response, Epc1 and Epc2 KD in both normal and leukemic cells induced γH2A.X. However, only in leukemia cells was a transcriptional signature of MYC activation noted together with accumulation of MYC protein, prior to induction of Bim and subsequent apoptosis. This observation reveals a novel mechanism operational in leukaemia cells but not normal myeloid cells by which protein levels of the critical cellular oncogene MYC are limited by EPC/EP400. This counterintuitive observation refines the paradigm for MYC in transformed cells: when the function of EPC and its associated EP400 complex components is inhibited, thus impairing the DNA damage response, MYC accumulates and sensitises cells to apoptotic death. Thus EPC1 and EPC2 are components of a complex which serves to prevent MYC accumulation in myeloid leukemia cells and their sensitisation to apoptosis, thus sustaining oncogenic potential. Therapeutic targeting of this complex may facilitate restoration of the natural tumour suppressor mechanisms that prevent cellular transformation by MYC and enhance chemosensitivity of AML cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Spliceosome Mutant Myeloid Malignancies Are Preferentially Sensitive to PARP Inhibition

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 322-322
Author(s):  
Dang Hai Nguyen ◽  
Zhiyan Silvia Liu ◽  
Sayantani Sinha ◽  
Maxwell Bannister ◽  
Erica Arriaga-Gomez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Dna Damage Response ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Splicing Factor ◽  
Leukemia Cells ◽  
Damage Response ◽  
Parp Activity ◽  
Mutant Cells

Abstract Somatic heterozygous mutations in spliceosome genes SRSF2, U2AF1, and SF3B1 commonly occur in patients with myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Moreover, SRSF2 and U2AF1 mutations associate with poor survival and high risk of progression to AML, representing a unique genetic vulnerability for targeted therapy. We and others previously found that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single-stranded DNA, are a source of genomic instability induced by different spliceosome mutants. We further showed that inhibition of ATR kinase activity preferentially kills spliceosome mutant cells in an R-loop-dependent manner. Inspired by ATR inhibition results, we performed a focused drug screen with inhibitors targeting additional DNA damage response pathways to identify novel therapeutic vulnerabilities generated by spliceosome mutations. We generated a murine leukemia model by overexpressing the MLL-AF9 fusion oncogene on an Srsf2 P95H/+background, a mutational combination that is found in ~10% of MLL-rearranged leukemias. Surprisingly, we found that Srsf2 P95H/+cells are more sensitive to five inhibitors targeting ADP-ribosyltransferases or PARP (olaparib, talazoparib, rucaparib, niraparib, veliparib) (Figs 1A-B). Olaparib (PARPi)-treated Srsf2 P95H/+cells exhibited increased apoptosis compared to Srsf2 +/+ cells as determined by AnnexinV (Fig 1C). PARPi sensitivity was also observed in isogenic murine MLL-AF9 U2af1 S34F/+cells compared to MLL-AF9 U2af1 +/+ cells (Fig 1D). These data highlight that both SRSF2 P95H and U2AF1 S34F mutations create a common vulnerability that is dependent on PARP activity for survival. To evaluate PARP activity in cells, we used isogenic K562 leukemia cells expressing SRSF2 P95H and U2AF1 S34F mutations from their endogenous loci and monitored PAR (poly(ADP-ribose)) chain levels, a marker of PARP activity. Both SRSF2 P95H and U2AF1 S34F cells exhibited elevated PAR levels compared to wildtype cells (Figs 1E-F). PARPi treatment significantly suppressed PAR signals in SRSF2 P95H and U2AF1 S34F cells. PARP inhibitors target both PARP1 and PARP2 enzymes, of which PARP1 plays a key role in DNA damage response. We used CRISPR-Cas9 to knockout PARP1 gene to determine the major PARP responsible for elevated PAR level in these leukemia cells. PARP1 deletion abrogated elevated PAR levels in U2AF1 S34F (Fig 1G) and SRSF2 P95H cells (data not shown). Altogether, we demonstrated that SRSF2 P95H and U2AF1 S34F cells trigger a PARP1 response critical for cell survival. To test whether increased PAR level arises from U2AF1 S34F-induced R-loops, we generated U2AF1 S34F cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within RNA:DNA hybrids. Induction of RNaseH1 in U2AF1 S34F cells significantly reduced PAR levels, showing that U2AF1 S34F-induced PAR chains is R-loop-dependent (Fig 1H). Moreover, RNaseH1 overexpression suppressed the growth inhibition of PARPi-treated U2AF1 S34F cells (Fig 1I). Collectively, these results suggest that U2AF1 S34F mutants induce R-loop accumulation and elicit an R-loop-associated PARP1 signaling to promote cell survival. We next tested whether combining ATR inhibitor (ATRi) can further exacerbate PARPi sensitivity in spliceosome mutant cells. To examine ATR activity, we monitored phosphorylated RPA (Replication Protein A, or pRPA), a known ATR substrate. pRPA level was enhanced in PARPi-treated SRSF2 P95H cells compared to PARPi-treated SRSF2 WT cells but was suppressed when treated with ATRi (Fig 1J), suggesting that splicing factor mutant cells are more reliant on ATR function in the context of PARPi. Importantly, the combination of PARPi with ATRi, but not with ATMi, significantly promoted cell growth inhibition in SRSF2 P95H cells compared to SRSF2 WT cells or to SRSF2 P95H cells treated with individual compounds alone (Fig 1K). Collectively, these data provide a pre-clinical rationale that splicing factor mutant leukemias are preferentially sensitive to PARP1 modulation compared to their wildtype counterpart. Moreover, combining PARPi and ATRi may further sensitize spliceosome mutant cells and could represent a new therapeutic strategy in myeloid leukemia patients harboring these mutations (Fig 1L). Figure 1 Figure 1. Disclosures Graubert: Calico: Research Funding; Janssen: Research Funding; astrazeneca: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document