Srinivas Chatla | Temple University (original) (raw)
Papers by Srinivas Chatla
Cancer Research, Nov 14, 2023
Poly (ADP-ribose) polymerase (PARP) inhibitors represent a promising new class of agents that hav... more 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 b...
Blood
Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosi... more Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/one-carbon cycle metabolism contributed to accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1]. To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLq) by ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLq and its proteasomal degradation. Overexpression of POLq in OTK-positive cells resulted in efficient repair of DPC-containing DNA double-strand breaks (DSBs) by POLq-mediated end-joining (TMEJ). Transforming activity of OTKs and other leukemia-inducing oncogenes, especially of those causing inhibition of BRCA1/2 -mediated homologous recombination (HR) with and without concomitant inhibition of DNA-PK -dependent non-homologous end-joining (D-NHEJ), was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLq polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitor or DPC-inducing drug etoposide enhanced anti-leukemia effect of POLq inhibitor (POLqi) in vitro and in vivo. In conclusion, we demonstrated that POLq plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde and that it can be targeted to achieve therapeutic effect.
Blood Cancer Journal
Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98... more 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 est...
The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that hav... more The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that have shown efficacy in treating various cancers, especially those harboring BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPi) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, PARPi resistance is common and develops through multiple mechanisms. Restoring HR and/or stabilizing replication forks are two major mechanisms of PARPi resistance in BRCA1/2-mutated cells. To further understand the mechanisms of drug resistance to PARPi, we took an unbiased approach with a CRISPR-pooled genome-wide library to screen new genes whose loss-of-function confers resistance to PARPi olaparib. We identified ZNF251, a transcription factor, and found that its loss-of-function led to the PARPi resistance in mult...
bioRxiv, 2022
The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that hav... more The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that have shown efficacy in treating various cancers, especially those harboring BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPi) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, PARPi resistance is common and develops through multiple mechanisms. Restoring HR and/or stabilizing replication forks are two major mechanisms of PARPi resistance in BRCA1/2-mutated cells. To further understand the mechanisms of drug resistance to PARPi, we took an unbiased approach with a CRISPR-pooled genome-wide library to screen new genes whose loss-of-function confers resistance to PARPi olaparib. We identified ZNF251, a transcription factor, and found that its loss-of-function led to the PARPi resistance in multiple BRCA1-mutated breast and ovarian cancer lines. Elevated activities of both HR and nonhomologous end joining (NHEJ) repair were detected in cancer cells harboring BRCA1 mutation and ZNF251 deletion (BRCA1mut+ZNF251del) and were associated with enhanced expression of RAD51 and Ku70/Ku80, respectively. Furthermore, we showed that a DNA-PKcs inhibitor restored sensitivity of BRCA1mut+ZNF251del cells to PARPi ex vivo and in vivo. Taken together, our study discovered a novel gene ZNF251 whose loss-of-function conferred resistance to PARPi in BRCA1-mutated breast and ovarian cancers and identified DNA repair pathway responsible for this effect.
Journal of Clinical Investigation, 2022
The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is crit... more The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.
Haematologica, 2022
The immune receptor TREM1 (Triggering receptor expressed on myeloid cells 1) is a master regulato... more The immune receptor TREM1 (Triggering receptor expressed on myeloid cells 1) is a master regulator of inflammatory response. Compelling evidence suggests important pathological roles for TREM1 in various types of solid tumors. However, the role of TREM1 in hematologic malignancies is not known. Our previous study demonstrates that TREM1 cooperates with diminished DNA damage response to induce expansion of pre-leukemic hematopoietic stem cells (HSCs) in mice deficient for the Fanconi anemia gene Fanca. Here we investigate TREM1 in leukemogenesis using mouse models of the DNA repair-deficient Fanca-/- and the oncogenic MLL-AF9 or KrasG12D. We found that Trem1 was highly expressed in pre-leukemic HSCs and leukemia stem cells (LSCs). By selective deletion of the Trem1 gene in the hematopoietic compartment, we showed that ablation of Trem1 reduced leukemogenic activity of the pre-leukemic HSCs and LSCs in mice. Trem1 was required for the proliferation of the pre-leukemic HSCs and LSCs. F...
Gene, Jan 28, 2017
Scriptaid (SCR), a well-known histone deacetylase inhibitor, cause various cellular effects such ... more Scriptaid (SCR), a well-known histone deacetylase inhibitor, cause various cellular effects such as cell growth inhibition and apoptosis. In this study, we have evaluated the anti-cancer effects of scriptaid in HeLa cells, IMR-32 and HepG2 cells. Scriptaid inhibited the growth of HeLa cells with IC50 of 2μM at 48h in a dose-dependent manner. Flow-cytometric analysis indicated that SCR induced apoptosis. Scriptaid was found to inhibit HDAC-8 effectively than other HDAC inhibitor such as TSA as observed by HDAC-8 assay, Western blotting and modelling study. This observation was further strengthened by an artificial neuronal network (ANN) model.
Apoptosis, 2016
In eukaryotes, transcriptional regulation occurs via chromatin remodeling, mainly through post tr... more In eukaryotes, transcriptional regulation occurs via chromatin remodeling, mainly through post translational modifications of histones that package DNA into structural units. Histone deacetylases (HDACs) are enzymes that play important role in various biological processes by repressing gene expression. Suberoylanilide hydroxamic acid (SAHA) is a known HDAC inhibitor that showed significant anti cancer activity by relieving gene silencing against hematologic and solid tumors. We have designed and synthesized a series of SAHA analogs C1-C4 and performed biological studies to elucidate its anti-cancer effects. It is observed that SAHA analogs significantly inhibited cell proliferation and induced apoptosis in hepatocellular carcinoma (HCC) cell lines HepG2 and SK-HEP-1. These analogs also showed non-toxic activity towards primary human hepatocytes, which describes its tumor specificity. SAHA analogs exhibited strong HDAC inhibition, which is 2-3 fold higher compared to SAHA. Moreover, these molecules induced hyper acetylation of histone H3 at various positions on the lysine residue. Further, it is observed that SAHA analogs are strong inducers of apoptosis, as they regulated the expression of various proteins involved in both extrinsic and intrinsic pathways. Interestingly, SAHA analogs induced upregulation of tumor suppressor miRNAs by activating its biogenesis pathway. Further, it is confirmed by microRNA (miRNA) prediction tools that these miRNAs are capable of targeting various anti-apoptotic genes. Based on these findings we conclude that SAHA analogs could be strong HDAC inhibitors with promising apoptosis inducing nature in HCC.
MedChemComm, 2013
General Procedures (Chemistry and Biology) 2-5 Fluorescent activated cell sorting (FACS) analysis... more General Procedures (Chemistry and Biology) 2-5 Fluorescent activated cell sorting (FACS) analysis of compounds 4a-ad 6 Cell viability in MCF-7 breast carcinoma cells 7 Cell cycle distribution Table 8 Senescence in K562 cells 9 Telomerase activity in A549 cancer cell lines 9 Spectral Data and Procedure of Compounds 7ac, 9, 10ac and 4aad 1028 Electronic Supplementary Material (ESI) for Medicinal Chemistry Communications This journal is © The Royal Society of Chemistry 2013 (c) Cell cycle analysis: 5×10 5 MCF-7 cells were seeded in 60 mm dish and were allowed to grow for 24 h. Compounds 4a-ad were added at a final concentration of 4 μM to the culture media, and the cells were incubated for an additional 24 h. Cells were harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 °C for 30 min, washed with PBS and incubated with 1 mg/ml RNase A solution (Sigma) at 37°C for 30 min. Cells were collected by centrifugation at 2000 rpm for 5 min and further stained with 250 μL of DNA staining solution [10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ water at room temperature for 30 min in the dark]. The DNA contents of 20,000 events were measured by flow cytometer (DAKO CYTOMATION, Beckman Coulter, Brea, CA). Histograms were analyzed using Summit Software. (d) BrdU cell proliferation assay: This assay was carried out by using the 5-Bromo-2deoxyuridine (BrdU) cell proliferation assay kit (Millipore) to assess the effect of compounds 4i, 4p, 4q, 4s, 4w and Doxo on proliferation of MCF-7 cells. 1 × 10 4 cells were seeded and allowed to grow for 24 h. BrdU was added and allowed to incorporate for 5 h followed by the addition of test compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4μM concentration for 24 h. Fixation was done for 30 min at room temperature. The cells were then washed, anti-BrdU antibody was added which binds to BrdU that was incorporated in the cells. After 1 h incubation, 100 μl anti-BrdU goat anti-mouse horse raddish peroxidise (HRP)-conjugated secondary antibody (1:2000) was added and incubated for 30 min. Washing procedures were followed according to the manufacturer's instructions. TMB substrate (100 μL) was added, incubated for another 30 min at room temperature and O.D values were taken at a wave length of 450 nm. Lower O.D values reflect lower BrdU concentrations in the sample and thus an indirect depiction of a low cell proliferation rate. (e) TRAPeze XL Telomerase assay: This detection kit (Millipore) is a sensitive as well as rapid PCR based fluorescent assay for detecting telomerase activity in cell extracts. Treatments were given for 24h with compounds 4i, 4p, 4q, 4s, 4w and Doxo at a concentration of 4 µM. Lysis of cells was carried out using CHAPS buffer. The extracts were further used for PCR reaction. Heat inactivated control untreated MCF-7 cell
ChemMedChem, 2010
A new class of imidazo[2,1-b]thiazole chalcone derivatives were synthesized and evaluated for the... more A new class of imidazo[2,1-b]thiazole chalcone derivatives were synthesized and evaluated for their anticancer activity. These chalcone derivatives show promising activity, with log GI(50) values ranging from -7.51 to -4.00. The detailed biological aspects of these derivatives toward the MCF-7 cell line were studied. Interestingly, these chalcone derivatives induced G(0)/G(1)-phase cell-cycle arrest, down-regulation of G(1)-phase cell-cycle regulatory proteins such as cyclin D1 and cyclin E1, and up-regulation of CDK4. Moreover, these compounds elicit the characteristic features of apoptosis such as enhancement in the levels of p53, p21, and p27, suppression of NF-κB, and up-regulation of caspase-9. One of these chalcone derivatives, 3 d, is potentially well suited for detailed biological studies, either alone or in combination with existing therapies.
Bioorganic & Medicinal Chemistry Letters, 2012
aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compou... more aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compounds were found to arrest cell cycle via a novel cross species microRNA-dependent regulatory pathway interpreting an unexpected link between cell cycle arrest and microRNA mediated control in cancer.
![Research paper thumbnail of Synthesis, DNA-binding ability and anticancer activity of benzothiazole/benzoxazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates](https://attachments.academia-assets.com/95398022/thumbnails/1.jpg)
](Bioorganic & Medicinal Chemistry, 2010
As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential antican... more As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.
Cancer Cell International, 2011
Background Hepatocellular carcinoma (HCC) accounts for majority of liver cancers and is the leadi... more Background Hepatocellular carcinoma (HCC) accounts for majority of liver cancers and is the leading cause of cancer related death in Asia. Like any other cancer, HCC develops when there is a mutation to the cellular machinery that causes the cell to replicate at a higher rate and results in the loss of apoptosis. Therefore, a delicate balance between the expression of various genes involved in proliferation and apoptosis decide the ultimate fate of the cell to undergo rapid proliferation (cancer) or cell death. Results The benzothiazole based compounds exhibited effective cytotoxicity at 4 μM concentration and have shown G1 cell cycle arrest with decrease in levels of G1 cell cycle proteins such as cyclin D1 and Skp2. Involvement of tumour suppressor proteins such as PTEN and p53 was studied. Interestingly these compounds displayed decrease in the phosphorylated forms of AKT, p38 MAPK and ERK1/2 which play a vital role in cell proliferation. Compounds have exhibited strong and signi...
Nature Communications, 2021
Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidenc... more Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidence that prolonged and intensive chemotherapy often fails to eradicate leukemic stem cells, which are protected by the bone marrow niche and can induce relapse. Thus, new therapeutic approaches to overcome chemoresistance are urgently needed. By conducting an ex vivo small molecule screen, here we have identified Quinacrine (QC) as a sensitizer for Cytarabine (AraC) in treating acute lymphoblastic leukemia (ALL). We show that QC enhances AraC-mediated killing of ALL cells, and subsequently abrogates AraC resistance both in vitro and in an ALL-xenograft model. However, while combo AraC+QC treatment prolongs the survival of primary transplanted recipients, the combination exhibits limited efficacy in secondary transplanted recipients, consistent with the survival of niche-protected leukemia stem cells. Introduction of Cdc42 Activity Specific Inhibitor, CASIN, enhances the eradication of ALL ...
Blood
The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSC... more The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a...
International Journal of Stem Cells, 2019
Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of c... more Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of cancer particularly leukemia. Here we show that inactivation of the non-homologous end-joining (NHEJ) activity of DNA-PKcs prevented DNA damage-induced expansion of FA pre-leukemic hematopoietic stem cells (HSCs). Furthermore, we performed serial BM transplantation to demonstrate that the DNA damage-induced expanded FA HSC compartment contained pre-leukemic stem cells that required the NHEJ activity of DNA-PKcs to induce leukemia in the secondary recipients. These results suggest that NHEJ may collaborate with FA deficiency to promote DNA damage-induced expansion of pre-leukemic HSCs.
Stem Cell Research, 2019
Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes inc... more Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.
Cancer Research, Nov 14, 2023
Poly (ADP-ribose) polymerase (PARP) inhibitors represent a promising new class of agents that hav... more 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 b...
Blood
Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosi... more Leukemia cells accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/one-carbon cycle metabolism contributed to accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1]. To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLq) by ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLq and its proteasomal degradation. Overexpression of POLq in OTK-positive cells resulted in efficient repair of DPC-containing DNA double-strand breaks (DSBs) by POLq-mediated end-joining (TMEJ). Transforming activity of OTKs and other leukemia-inducing oncogenes, especially of those causing inhibition of BRCA1/2 -mediated homologous recombination (HR) with and without concomitant inhibition of DNA-PK -dependent non-homologous end-joining (D-NHEJ), was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLq polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitor or DPC-inducing drug etoposide enhanced anti-leukemia effect of POLq inhibitor (POLqi) in vitro and in vivo. In conclusion, we demonstrated that POLq plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde and that it can be targeted to achieve therapeutic effect.
Blood Cancer Journal
Deletion of ABL1 was detected in a cohort of hematologic malignancies carrying AML1-ETO and NUP98... more 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 est...
The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that hav... more The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that have shown efficacy in treating various cancers, especially those harboring BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPi) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, PARPi resistance is common and develops through multiple mechanisms. Restoring HR and/or stabilizing replication forks are two major mechanisms of PARPi resistance in BRCA1/2-mutated cells. To further understand the mechanisms of drug resistance to PARPi, we took an unbiased approach with a CRISPR-pooled genome-wide library to screen new genes whose loss-of-function confers resistance to PARPi olaparib. We identified ZNF251, a transcription factor, and found that its loss-of-function led to the PARPi resistance in mult...
bioRxiv, 2022
The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that hav... more The poly (ADP-ribose) polymerases (PARPs) inhibitors are an exciting new class of agents that have shown efficacy in treating various cancers, especially those harboring BRCA1/2 mutations. The cancer associated BRCA1/2 mutations disrupt DNA double strand break (DSB) repair by homologous recombination (HR). PARP inhibitors (PARPi) have been applied to trigger synthetic lethality in BRCA1/2-mutated cancer cells by promoting the accumulation of toxic DSBs. Unfortunately, PARPi resistance is common and develops through multiple mechanisms. Restoring HR and/or stabilizing replication forks are two major mechanisms of PARPi resistance in BRCA1/2-mutated cells. To further understand the mechanisms of drug resistance to PARPi, we took an unbiased approach with a CRISPR-pooled genome-wide library to screen new genes whose loss-of-function confers resistance to PARPi olaparib. We identified ZNF251, a transcription factor, and found that its loss-of-function led to the PARPi resistance in multiple BRCA1-mutated breast and ovarian cancer lines. Elevated activities of both HR and nonhomologous end joining (NHEJ) repair were detected in cancer cells harboring BRCA1 mutation and ZNF251 deletion (BRCA1mut+ZNF251del) and were associated with enhanced expression of RAD51 and Ku70/Ku80, respectively. Furthermore, we showed that a DNA-PKcs inhibitor restored sensitivity of BRCA1mut+ZNF251del cells to PARPi ex vivo and in vivo. Taken together, our study discovered a novel gene ZNF251 whose loss-of-function conferred resistance to PARPi in BRCA1-mutated breast and ovarian cancers and identified DNA repair pathway responsible for this effect.
Journal of Clinical Investigation, 2022
The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is crit... more The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.
Haematologica, 2022
The immune receptor TREM1 (Triggering receptor expressed on myeloid cells 1) is a master regulato... more The immune receptor TREM1 (Triggering receptor expressed on myeloid cells 1) is a master regulator of inflammatory response. Compelling evidence suggests important pathological roles for TREM1 in various types of solid tumors. However, the role of TREM1 in hematologic malignancies is not known. Our previous study demonstrates that TREM1 cooperates with diminished DNA damage response to induce expansion of pre-leukemic hematopoietic stem cells (HSCs) in mice deficient for the Fanconi anemia gene Fanca. Here we investigate TREM1 in leukemogenesis using mouse models of the DNA repair-deficient Fanca-/- and the oncogenic MLL-AF9 or KrasG12D. We found that Trem1 was highly expressed in pre-leukemic HSCs and leukemia stem cells (LSCs). By selective deletion of the Trem1 gene in the hematopoietic compartment, we showed that ablation of Trem1 reduced leukemogenic activity of the pre-leukemic HSCs and LSCs in mice. Trem1 was required for the proliferation of the pre-leukemic HSCs and LSCs. F...
Gene, Jan 28, 2017
Scriptaid (SCR), a well-known histone deacetylase inhibitor, cause various cellular effects such ... more Scriptaid (SCR), a well-known histone deacetylase inhibitor, cause various cellular effects such as cell growth inhibition and apoptosis. In this study, we have evaluated the anti-cancer effects of scriptaid in HeLa cells, IMR-32 and HepG2 cells. Scriptaid inhibited the growth of HeLa cells with IC50 of 2μM at 48h in a dose-dependent manner. Flow-cytometric analysis indicated that SCR induced apoptosis. Scriptaid was found to inhibit HDAC-8 effectively than other HDAC inhibitor such as TSA as observed by HDAC-8 assay, Western blotting and modelling study. This observation was further strengthened by an artificial neuronal network (ANN) model.
Apoptosis, 2016
In eukaryotes, transcriptional regulation occurs via chromatin remodeling, mainly through post tr... more In eukaryotes, transcriptional regulation occurs via chromatin remodeling, mainly through post translational modifications of histones that package DNA into structural units. Histone deacetylases (HDACs) are enzymes that play important role in various biological processes by repressing gene expression. Suberoylanilide hydroxamic acid (SAHA) is a known HDAC inhibitor that showed significant anti cancer activity by relieving gene silencing against hematologic and solid tumors. We have designed and synthesized a series of SAHA analogs C1-C4 and performed biological studies to elucidate its anti-cancer effects. It is observed that SAHA analogs significantly inhibited cell proliferation and induced apoptosis in hepatocellular carcinoma (HCC) cell lines HepG2 and SK-HEP-1. These analogs also showed non-toxic activity towards primary human hepatocytes, which describes its tumor specificity. SAHA analogs exhibited strong HDAC inhibition, which is 2-3 fold higher compared to SAHA. Moreover, these molecules induced hyper acetylation of histone H3 at various positions on the lysine residue. Further, it is observed that SAHA analogs are strong inducers of apoptosis, as they regulated the expression of various proteins involved in both extrinsic and intrinsic pathways. Interestingly, SAHA analogs induced upregulation of tumor suppressor miRNAs by activating its biogenesis pathway. Further, it is confirmed by microRNA (miRNA) prediction tools that these miRNAs are capable of targeting various anti-apoptotic genes. Based on these findings we conclude that SAHA analogs could be strong HDAC inhibitors with promising apoptosis inducing nature in HCC.
MedChemComm, 2013
General Procedures (Chemistry and Biology) 2-5 Fluorescent activated cell sorting (FACS) analysis... more General Procedures (Chemistry and Biology) 2-5 Fluorescent activated cell sorting (FACS) analysis of compounds 4a-ad 6 Cell viability in MCF-7 breast carcinoma cells 7 Cell cycle distribution Table 8 Senescence in K562 cells 9 Telomerase activity in A549 cancer cell lines 9 Spectral Data and Procedure of Compounds 7ac, 9, 10ac and 4aad 1028 Electronic Supplementary Material (ESI) for Medicinal Chemistry Communications This journal is © The Royal Society of Chemistry 2013 (c) Cell cycle analysis: 5×10 5 MCF-7 cells were seeded in 60 mm dish and were allowed to grow for 24 h. Compounds 4a-ad were added at a final concentration of 4 μM to the culture media, and the cells were incubated for an additional 24 h. Cells were harvested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 °C for 30 min, washed with PBS and incubated with 1 mg/ml RNase A solution (Sigma) at 37°C for 30 min. Cells were collected by centrifugation at 2000 rpm for 5 min and further stained with 250 μL of DNA staining solution [10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and 0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ water at room temperature for 30 min in the dark]. The DNA contents of 20,000 events were measured by flow cytometer (DAKO CYTOMATION, Beckman Coulter, Brea, CA). Histograms were analyzed using Summit Software. (d) BrdU cell proliferation assay: This assay was carried out by using the 5-Bromo-2deoxyuridine (BrdU) cell proliferation assay kit (Millipore) to assess the effect of compounds 4i, 4p, 4q, 4s, 4w and Doxo on proliferation of MCF-7 cells. 1 × 10 4 cells were seeded and allowed to grow for 24 h. BrdU was added and allowed to incorporate for 5 h followed by the addition of test compounds 4i, 4p, 4q, 4s, 4w and Doxo at 4μM concentration for 24 h. Fixation was done for 30 min at room temperature. The cells were then washed, anti-BrdU antibody was added which binds to BrdU that was incorporated in the cells. After 1 h incubation, 100 μl anti-BrdU goat anti-mouse horse raddish peroxidise (HRP)-conjugated secondary antibody (1:2000) was added and incubated for 30 min. Washing procedures were followed according to the manufacturer's instructions. TMB substrate (100 μL) was added, incubated for another 30 min at room temperature and O.D values were taken at a wave length of 450 nm. Lower O.D values reflect lower BrdU concentrations in the sample and thus an indirect depiction of a low cell proliferation rate. (e) TRAPeze XL Telomerase assay: This detection kit (Millipore) is a sensitive as well as rapid PCR based fluorescent assay for detecting telomerase activity in cell extracts. Treatments were given for 24h with compounds 4i, 4p, 4q, 4s, 4w and Doxo at a concentration of 4 µM. Lysis of cells was carried out using CHAPS buffer. The extracts were further used for PCR reaction. Heat inactivated control untreated MCF-7 cell
ChemMedChem, 2010
A new class of imidazo[2,1-b]thiazole chalcone derivatives were synthesized and evaluated for the... more A new class of imidazo[2,1-b]thiazole chalcone derivatives were synthesized and evaluated for their anticancer activity. These chalcone derivatives show promising activity, with log GI(50) values ranging from -7.51 to -4.00. The detailed biological aspects of these derivatives toward the MCF-7 cell line were studied. Interestingly, these chalcone derivatives induced G(0)/G(1)-phase cell-cycle arrest, down-regulation of G(1)-phase cell-cycle regulatory proteins such as cyclin D1 and cyclin E1, and up-regulation of CDK4. Moreover, these compounds elicit the characteristic features of apoptosis such as enhancement in the levels of p53, p21, and p27, suppression of NF-κB, and up-regulation of caspase-9. One of these chalcone derivatives, 3 d, is potentially well suited for detailed biological studies, either alone or in combination with existing therapies.
Bioorganic & Medicinal Chemistry Letters, 2012
aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compou... more aza-Flavanones have been identified as a new class of selective microRNA inhibitors. These compounds were found to arrest cell cycle via a novel cross species microRNA-dependent regulatory pathway interpreting an unexpected link between cell cycle arrest and microRNA mediated control in cancer.
Bioorganic & Medicinal Chemistry, 2010
As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential antican... more As a continuation of our efforts to develop the benzimidazole-PBD conjugates as potential anticancer agents, a series of heteroaryl substituted benzimidazole linked PBD conjugates has been synthesized and evaluated for their anticancer potential in 60 human cancer cell lines. Most of the compounds exhibited promising anticancer activity and interestingly, compounds 4c and 4d displayed significant activity in most of the cell lines tested. Whereas, compound 4e showed selectivity in renal cancer cells with GI50 values of <10 and 70 nM against RXF 393 and UO-31 cell lines, respectively. Further, these compounds also showed significant DNA-binding affinity by thermal denaturation study using duplex form of calf thymus (CT) DNA.
Cancer Cell International, 2011
Background Hepatocellular carcinoma (HCC) accounts for majority of liver cancers and is the leadi... more Background Hepatocellular carcinoma (HCC) accounts for majority of liver cancers and is the leading cause of cancer related death in Asia. Like any other cancer, HCC develops when there is a mutation to the cellular machinery that causes the cell to replicate at a higher rate and results in the loss of apoptosis. Therefore, a delicate balance between the expression of various genes involved in proliferation and apoptosis decide the ultimate fate of the cell to undergo rapid proliferation (cancer) or cell death. Results The benzothiazole based compounds exhibited effective cytotoxicity at 4 μM concentration and have shown G1 cell cycle arrest with decrease in levels of G1 cell cycle proteins such as cyclin D1 and Skp2. Involvement of tumour suppressor proteins such as PTEN and p53 was studied. Interestingly these compounds displayed decrease in the phosphorylated forms of AKT, p38 MAPK and ERK1/2 which play a vital role in cell proliferation. Compounds have exhibited strong and signi...
Nature Communications, 2021
Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidenc... more Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidence that prolonged and intensive chemotherapy often fails to eradicate leukemic stem cells, which are protected by the bone marrow niche and can induce relapse. Thus, new therapeutic approaches to overcome chemoresistance are urgently needed. By conducting an ex vivo small molecule screen, here we have identified Quinacrine (QC) as a sensitizer for Cytarabine (AraC) in treating acute lymphoblastic leukemia (ALL). We show that QC enhances AraC-mediated killing of ALL cells, and subsequently abrogates AraC resistance both in vitro and in an ALL-xenograft model. However, while combo AraC+QC treatment prolongs the survival of primary transplanted recipients, the combination exhibits limited efficacy in secondary transplanted recipients, consistent with the survival of niche-protected leukemia stem cells. Introduction of Cdc42 Activity Specific Inhibitor, CASIN, enhances the eradication of ALL ...
Blood
The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSC... more The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a...
International Journal of Stem Cells, 2019
Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of c... more Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and high risk of cancer particularly leukemia. Here we show that inactivation of the non-homologous end-joining (NHEJ) activity of DNA-PKcs prevented DNA damage-induced expansion of FA pre-leukemic hematopoietic stem cells (HSCs). Furthermore, we performed serial BM transplantation to demonstrate that the DNA damage-induced expanded FA HSC compartment contained pre-leukemic stem cells that required the NHEJ activity of DNA-PKcs to induce leukemia in the secondary recipients. These results suggest that NHEJ may collaborate with FA deficiency to promote DNA damage-induced expansion of pre-leukemic HSCs.
Stem Cell Research, 2019
Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes inc... more Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.