Research in
Prof. Sathees C. Raghavan’s Laboratory

Mechanism of chromosomal translocations in leukemia and lymphoma

Research from our laboratory has identified the role of G-quadruplex DNA, one of the non-B DNA structures, in the generation of chromosomal translocations. We reported existence of one or more G-quadruplex structures at or near BCL2 major breakpoint region, and HOX11 breakpoint regions, which can explain the reported fragility during t(14;18) in follicular lymphoma (Nucleic Acids Res, 2011a, b) and t(10;14) translocations in T-cell acute lymphoblastic leukemia (Mol Cell Biol, 2013). Besides we observed that 40-50% of translocation breakpoint regions possess G-quadruplex forming motifs in leukemia and lymphoma patients (Genomics, 2012). Formation of G-quadruplex DNA structures has also been seen in case of BCL6 translocation breakpoint region associated with diffuse large B cell lymphoma, BCR fragile region in chronic myelogenous leukemia and BCL11b exon 4 seen in T cell leukemias (Unpublished). We have also identified transcription dependent R loop formation at c-MYC fragile region seen in Burkitt’s lymphoma (Unpublished). In a different study, we have also identified a unique RAG dependent mechanism by which BCL2 minor breakpoint cluster breaks during t(14;18) translocation seen in follicular lymphoma (J. Biol. Chem., 2012).In an interesting observation, we found that formation of G-quadruplex structures on DNA can protect it against ionizing radiation in vitro and inside cells (iScience, 2019).Our studies have determined the prevalence of the t(14;18) in the healthy Indian population (Annals in Hematology, 2010). The detailed understanding of the evolution of such cells in healthy individuals will be the key in elucidating the mechanism of lymphomagenesis, once a B-cell acquires a translocation (Cell Mol Life Sciences, 2012).

Regulation of RAGs in different cell types and its role in genomic instability

Studies from our laboratory revealed that RAG cleavage on heteroduplex DNA is preferred when cytosines are present at single-stranded regions (J Biol Chem, 2010). We observed that nonamer binding domain of RAG1 does not play a role, when it acts as a structure-specific nuclease (J Mol Biol, 2012, Scientific Reports, 2016). Very recently, we observed that ZnC2 (previously termed as ZFB) of RAG1 plays a critical role, when RAGs act as a structure-specific nuclease (Biochemical J, 2020). Further, we observed that regulation of this property of RAG is dependent on the length, sequence, and phasing of the double-stranded flanking region (FEBS J., 2015). A V(D)J recombination nonamer when present next to heteroduplex DNA can improve the efficiency of RAG-mediated cleavage on non B-DNA structures (Biochem J., 2012). In collaboration with others, we observed that methylated CpGs play a significant role in the generation of chromosomal translocations (Cell, 2008). More importantly, we observed that several chromosomal translocation breakpoint junctions also possess a nonamer sequence, at or near the breakpoint region that possesses CpGs, thus explaining the role of both DNA structures and sequence in genome stability associated with cancer (unpublished data). In an independent study, we have identified two novel miRNAs that have the ability to regulate expression of RAGs in B cell developmental stage-specific manner (unpublished data). These findings provide novel insights into the regulation of the pathological and physiological functions of RAGs. In an interesting study, we also investigated the impact of clinically used HIV inhibitors on the immune system in mouse models by demonstrating its impact on RAG functions in host cells (Cell Death Dis, 2017, Cell Death Discovery, 2020).

Mechanism of DNA double-strand break repair and its regulation in

normal tissues and cancer cells

Using a biochemical assay system established in the laboratory, we showed that testicular cells possessed a highly efficient NHEJ (Cell Mol Life Sci, 2011). Among somatic tissues, lungs exhibit the highest end joining activity followed by spleen and brain (Cell Mol Life Sci, 2011). We find that cancer cells expressing higher level of BCL2 possess lower NHEJ as BCL2 can interact with KU, the DNA end binding proteins (J Biol Chem, 2010). We have also established an in vitro cell-free assay system to investigate a sub-pathway of NHEJ known as microhomology-mediated end joining (MMEJ) and showed that MMEJ can operate in normal cells even when classical NHEJ is present (Cell Death Dis, 2015). We also observed that MMEJ is upregulated in cancers such as diffuse large B cell lymphoma (Molecular Carcinogenesis, 2019). Our studies have also shown that classical NHEJ is the predominant mechanism of repair in the early stages of mouse embryonic development, while MMEJ dominates in the later stages (J Mol Biol, 2012). Besides, our studies for the first time showed that Shrimp (Penaeus monodon) does possess an efficient DSB repair system, although, unlike higher order organisms, classical-NHEJ is undetectable (DNA Research, 2017).Further, our studies revealed that MMEJ mediated DSB repair in mitochondrial DNA can explain the mitochondrial DNA deletions observed in several human diseases associated with mitochondrial genome instability in a Ligase III dependent manner (Mol Biol Cell, 2015). In contrast, maintenance of mitochondrial DNA integrity is dependent on homologous recombination, when double-strand breaks are induced (Cell Mol Life Sci, 2017). We also discovered an additional novel function of Endonuclease G in mitochondria (unpublished).Using a variety of approaches, we demonstrated that coupled with pathophysiological changes, Endosulfan, a pesticide used in India and other Asian countries, induced testicular cell death. It affected spermatogenesis leading to reduction in sperm quality and quantity, thereby resulting in male infertility (Cell Death Discovery, 2015; Cell Death Dis., 2015; J. Bioscience, 2017). Treatment with Endosulfan resulted in induction of ROS, leading to DNA strand breaks, altered DNA damage response leading to enhanced level of microhomology mediated NHEJ, thus leading to elevated genome rearrangements and deletions (Carcinogenesis, 2016).

Cancer therapeutics

In an important finding, we identified a small molecule inhibitor, SCR7 that can abrogate NHEJ, one of the DSB repair pathways, both in vitro and ex vivo in a Ligase IV dependent manner (Cell, 2012). Multiple reports have revealed that SCR7 can improve efficiency of precise genome editing by CRISPR/Cas9 (Chemistry & Biology, 2015; FEBS J, 2015). Recent studies have revealed that SCR7 can be present in multiple chemical forms and still can inhibit NHEJ efficiently in a Ligase IV dependent manner (FEBS J, 2018). An encapsulated version of SCR7 exhibited five times more efficiency than its parental form (Faraday Discussions, 2015; Macromol Biosci, 2015). Dose titration revealed that SCR7 can bring down the effective dose of gamma-radiation by 50-75% during radiotherapy (unpublished data). More recently, we identified three improved versions of parental SCR7 that exhibit 3-100 fold better efficacy with respect to induction of cytotoxicity in cancer cell lines and tumor regression in animal models (Molecular Carcinogenesis, 2020 and unpublished).Recently, we also reported the identification of a specific BCL2 inhibitor, Disarib (FEBS J, 2016; Biochemi Biophy Acta Rev Cancer, 2017) that induced cytotoxicity in cancer cells. Disarib inhibited tumor progression in multiple animal models without causing significant side effects (Biochem Pharmacol, 2016). Interestingly, head to head comparison of Disarib and ABT199 (the only FDA approved BCL2 inhibitor) revealed that Disarib is more efficient in the used experimental model systems (Biochem Pharmacol, 2017). Disarib is patented in India and preclinical studies using Disarib are in progress. Initial evaluation as per CDSCO guidelines reveal that Disarib does not show toxicity in mice and rats (Scientific Reports, 2020 and unpublished). Thus, the novel BCL2 inhibitor, identified in our study is a potentially promising molecule for clinical studies against BCL2 'high' cancers such as Chronic lymphocytic leukemia, acute myeloid leukemia, breast cancer, prostate cancer, etc. as it is target-specific and spares normal cells.In addition to this, various experiments were conducted to identify several important small molecule inhibitors against PARP, Topoisomerase II, Tubulin etc (Biochemical Pharmacology, 2009, PLoS One. 2013; Molecular Carcinogenesis, 2013; Bioorg Med Chem Lett. 2014; Targeted Oncology, 2016; Sci Rep. 2017). Besides, extracts and purified compounds prepared from several medicinal plants and fruits were shown for their potential role in cancer therapy (FEBS Letters, 2008; PLoS One, 2012; Sci. Rep., 2014; Sci Rep. 2016a; Sci Rep. 2016b).

Highlights of major discoveries
from the laboratory

Identification of first NHEJ inhibitor with a potential in cancer therapy and genome editing.

Characterization of a BCL2-specific inhibitor with a prospect to be developed as a chemotherapeutic drug.

Understanding mechanism and regulation of classical nonhomologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) in repair of double-strand break (DSB) in both nuclear and mitochondrial genome.

Understanding the role of DNA structure, and identification and characterization of factors, responsible for generation of chromosomal translocations in various cancers, such as leukemia and lymphoma.

Identification and biochemical characterization of nonstandard functions of RAG complex, the protein responsible for generation of diversity in immune system, both in lymphoid and nonlymphoid cells.

Selected Research Articles

Vidya Gopalakrishnan/ Sathees C. Raghavan, Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma. (2024) Cell Mol Life Sci. 10;81(1):21.

Ujjayinee Ray/ Identification and characterization of mercaptopyrimidine-based small molecules as inhibitors of nonhomologous DNA end joining. (2023) FEBS Journal 290(3):796-820.

Nitu Kumari/ Sathees C. Raghavan, Deficiency of Ligase IV Leads to Reduced NHEJ, Accumulation of DNA Damage, and can Sensitize Cells to Cancer Therapeutics. (2023) Genomics 115(6):110731.

Rupa Kumari/ Urbi Roy/ Sathees C. Raghavan, MicroRNA, miR-501 regulate the V(D)J recombination in B cells. (2023) Biochemical Journal 480(24):2061-2077.

Nitu Kumari/ Kohal Das/ Shivangi Sharma/ Sathees C. Raghavan, Evaluation of the potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt's lymphoma (2023) J Biological Chemistry 299(12):105431.

Sumedha Dahal/ Sathees C Raghavan, Unleashing a novel function of endonuclease G in mitochondrial genome instability (2022) Elife. 2022;11:e69916.

Amita M Paranjape/ Sagar S Desai/ Sathees C Raghavan, Nonamer dependent RAG cleavage at CpGs can explain mechanism of chromosomal translocations associated to lymphoid cancers. (2022) PLoS Genetics. 18(10):e1010421.

Rupa Kumari/ Urbi Roy/ Sathees C Raghavan, MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development (2021) Cell Reports, 36 (2):109390.

Nitu Kumari/ Supriya S Vartak/ Sumedha Dahal / Sathees C Raghavan, G-quadruplex Structures Contribute to Differential Radiosensitivity of the Human Genome (2019) iScience 21:288-307.

Sumedha Dahal/ Sathees C. Raghavan, Homologous Recombination Mediated Repair of DNA Double-strand Breaks Operates in Mammalian Mitochondria (2018) Cell Mol Life Sci. 75(9):1641-1655.

Vivek Tripathi/ Sathees C. Raghavan and Sagar Sengupta, MRN complex-dependent recruitment of ubiquitylated BLM helicase to DSBs negatively regulates DNA repair pathways (2018) Nature Communication 9:10.

Divyaanka Iyer/ Sathees C. Raghavan, Identification of a Novel BCL2 Specific Inhibitor that Binds Predominantly to its BH1 Domain (2016) FEBS Journal 283:3408-37.

Satish Kumar Tadi/ Sathees C. Raghavan , Microhomology Mediated End Joining is the Principal Mediator of Double-strand Break Repair During Mitochondrial DNA Lesions (2016) Mol Biol Cell. 27: 223-35.

Robin Sebastian and Sathees C. Raghavan Induction of DNA Damage and Erroneous Repair can Explain Genomic Instability Caused by Endosulfan (2016) Carcinogenesis 37:929-40.

Sheetal Sharma/ Sathees C. Raghavan, Homology and Enzymatic Requirements of Microhomology Dependent Alternative End Joining (2015) Cell Death Dis. 6: e.1697.

Mridula Nambiar/ Sathees C. Raghavan G-quadruplex Structures Formed at the HOX11 Breakpoint Region contribute to its fragility during t(10;14) Translocation in T-cell Leukemia (2013) Mol Cell Biol. 33: 4266-81.

Mrinal Srivastava/ Sathees C. Raghavan, An Inhibitor of Nonhomologous Endjoining Abrogates Double-strand Break Repair and Impedes Cancer Progression (2012) Cell 151:1474-87.

Mayilaadumveettil Nishana and Sathees C. Raghavan, A V(D)J recombination nonamer can regulate RAG cleavage at non-B DNA structures: implications in genomic instability and cancer (2012) Biochemical Journal (Disease) 448:115-125.

Mridula Nambiar/ Sathees C. Raghavan, Characterization of G-quadruplex Structure at BCL2 Major Breakpoint Region of t(14;18) translocation in follicular lymphoma (2011) Nucleic Acids Res. 39:936-48.

Sheetal Sharma/ Sathees C. Raghavan, Nonhomologous DNA End Joining is Most Efficient in Lungs among Somatic Tissues, and is Equivalent to Germ Cells (2011) Cell Mol Life Sci. 68:661-76.

Tadi Satish Kumar/ Sathees C. Raghavan, Anti-apoptotic Protein, BCL2, Downregulates Nonhomologous DNA End Joining in Cancer Cells (2010) J Biological Chemistry 285:32657-32670.

Abani Kanta Naik/ Sathees C. Raghavan Cytosines, but not purines, determine RAG induced breaks on heteroduplex DNA Structures: Implications for genomic instability (2010) J Biological Chemistry 285: 7587-97.

Albert Tsai/ Sathees C. Raghavan/ Michael R. Lieber, Human Chromosomal Translocations at CpG Sites and a Theoretical Basis for their Lineage and Stage Specificity (2008) Cell 135: 1130-1142.

Sathees C Raghavan/ Michael R Lieber, A non-B DNA structure in the bcl-2 major break region is cleaved by RAGs (2004) Nature 428 88-93.

Publication of Latest Findings

Manthra Raveendran/ Shivangi Sharma/ Sathees C. Raghavan, A novel approach toward the multigram synthesis of a novel Bcl2-specific inhibitor, and evaluation of its biological activity. (2024) European Journal of Medicinal Chemistry Reports (In press).

Nitu Kumari/ Sathees C. Raghavan, Deficiency of Ligase IV Leads to Reduced NHEJ, Accumulation of DNA Damage, and can Sensitize Cells to Cancer Therapeutics. (2023) Genomics 115(6):110731.

Rupa Kumari/ Urbi Roy/ Sathees C. Raghavan, MicroRNA, miR-501 regulates the V(D)J recombination in B cells. (2023) Biochemical Journal 480(24):2061-2077.

Ujjayinee Ray, Vindya K Gopinatha, Shivangi Sharma, Laijau Goyary, Bibha Choudhary, Kempegowda Mantelingu, Kanchugarakoppal S Rangappa and Sathees C Raghavan, Identification and characterization of mercaptopyrimidine-based small molecules as inhibitors of nonhomologous DNA end joining (2023) FEBS J. doi: 10.1111/febs.16615. PMID: 36048168.

Sumedha Dahal, Humaira Siddiqua, Shivangi Sharma, Ravi K Babu, Diksha Rathore, Sheetal Sharma and Sathees C Raghavan, Unleashing a novel function of endonuclease G in mitochondrial genome instability (2022) Elife. 2022;11:e69916.

Anju Sharma, Arigesh Kaninathan, Sumedha Dahal, Susmita Kumari, Bibha Choudhary and Sathees C Raghavan, Exposure to Endosulfan can Cause Long Term Effects on General Biology, Including the Reproductive System of Mice (2022) Frontiers in Genetics Nov 24;13:1047746. doi: 10.3389/fgene.2022.1047746. eCollection 2022.PMID: 36506329.

Dipayan Ghosh, Susmita Kumari, Sathees C Raghavan, Depletion of RNASEH2 activity leads to accumulation of DNA double-strand breaks and reduced cellular survivability in T cell leukemia (2022) J Mol Biol. 2022 434(12):167617.

Amita M Paranjape, Sagar S Desai, Mayilaadumveettil Nishana, Urbi Roy, Namrata M Nilavar, Amrita Mondal, Rupa Kumari, Gudapureddy Radha, Vijeth Kumar Katapadi, Bibha Choudhary and Sathees C Raghavan, Nonamer dependent RAG cleavage at CpGs can explain mechanism of chromosomal translocations associated to lymphoid cancers (2022) PLoS Genetics. 18(10):e1010421.

Sumedha Dahal, Humaira Siddiqua, Vijeth K Katapadi, Divyaanka Iyer, Sathees C Raghavan, Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability (2022) FEBS Journal, 289(1):163-82.

Sagar S Desai, S Whadgar, Sathees C Raghavan, Bibha Choudhary, MiRAGDB: a knowledgebase of RAG regulators (2022) Frontiers in Immunology, 13:863110.

Rupa Kumari*, Urbi Roy*, Sagar S Desai, Namrata M Nilavar, Van Nieuwenhuijze, Amita Paranjape, Radha Gudapureddy, P Bawa, Mrinal Srivastava, Mridula Nambiar, KN Balaji, Adrian Liston, Bibha Choudhary, Sathees C Raghavan (*equals first authors), MicroRNA miR-29c regulates RAG1 expression and modulates V(D)J recombination during B cell development (2021) Cell Reports, 36 (2):109390.

Dipayan Ghosh, Sathees C Raghavan, Nonhomologous end joining: new accessory factors fine tune the machinery (2021) Trends in Genetics, 37(6):582-599.

Vidya Gopalakrishnan, Shivangi Sharma, Ujjayinee Ray, Meghana Manjunath, Divya Lakshmanan, Supriya S Vartak, Vindya K Gopinatha, Mrinal Srivastava, Mantelingu Kempegowda, Bibha Choudhary, Sathees C Raghavan, SCR7, an inhibitor of NHEJ can sensitize tumor cells to ionization radiation (2021) Molecular Carcinogenesis, 60 (9):627-43.

Most Cited Articles

Shikha Srivastava/ Sathees C. Raghavan, Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis (2016) Scientific Report 6: 24049. Citations 426

Mrinal Srivastava/ Sathees C. Raghavan, An Inhibitor of Nonhomologous Endjoining Abrogates Double-strand Break Repair and Impedes Cancer Progression (2012) Cell 151:1474-87. Citations 385

Albert Tsai/ Sathees C. Raghavan/ Michael R. Lieber, Human Chromosomal Translocations at CpG Sites and a Theoretical Basis for their Lineage and Stage Specificity (2008) Cell 135: 1130-1142. Citations 270

Sathees C Raghavan/ Michael R Lieber A non-B DNA structure in the bcl-2 major break region is cleaved by RAGs (2004) Nature 428 88-93. Citations 269

Mridula Nambiar, Vijayalakshmi Kari and Sathees C. Raghavan, Chromosomal translocations in cancer (2008) Biochim. Biophys. Acta, Reviews on Cancer, 1786:139-152. Citations 220

Mrinal Srivastava and Sathees C. Raghavan, DNA repair inhibitors in Cancer therapeutics

(2015) Chemistry & Biology. 22: 17-29. Citations 210

Kavitha C.V./ Sathees C. Raghavan Novel Derivatives of Spirohydantoin Induce Growth Inhibition Followed by Apoptosis in Leukemia Cells (2009) Biochemical Pharmacology 77:348-363. Citations 166

Sheetal Sharma/ Sathees C. Raghavan Homology and Enzymatic Requirements of Microhomology Dependent Alternative End Joining (2015) Cell Death and Diseases 6: e.1697. Citations 162

Mridula Nambiar and Sathees C. Raghavan, How DNA Breaks During Chromosomal Translocations in Cancer? (2011) Nucleic Acids Res. 39:5813-25. Citations 149

Mridula Nambiar/ Sathees C. Raghavan Formation of G-quadruplex Structure at BCL2 Major Breakpoint Region of t(14;18) translocation in follicular lymphoma (2011) Nucleic Acids Research 39:936-48, Citations 128

SS Karki/Sathees C. Raghavan Synthesis and biological evaluation of novel 2-aralkyl-5-substituted-6-(4′-fluorophenyl)-imidazo [2, 1-b][1, 3, 4] thiadiazole derivatives as potent anticancer agents (2011), European journal of medicinal chemistry 46 (6), 2109-2116, Citations 124

Supriya V Vartak/ Sathees C. Raghavan, Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing (2015) The FEBS journal 282 (22), 4289-4294, Citations 123

G Radha/ Sathees C. Raghavan BCL2: A promising cancer therapeutic target (2017) Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1868 (1), 309-314, Citations 118

Satish Kumar Tadi/ Sathees C. Raghavan, Microhomology Mediated End Joining is the Principal Mediator of Double-strand Break Repair During Mitochondrial DNA Lesions (2016) Molecular Biology of the Cell 27: 223-35. Citations 117

Kishore K.Chiruvella and SC Raghavan,Phytochemical and antimicrobial studies of methyl angolensate and luteolin-7-O-glucoside isolated from callus cultures of Soymida febrifuga (2007) International journal of biomedical science: IJBS 3 (4), 269, Citations 113

Mayilaadumveettil Nishana and Sathees C. Raghavan, Role of RAGs in the generation of antigen receptor diversity and beyond (2012) Immunology 137:271-81. Citations 97

Contact Us

FA-10, Department of Biochemistry
Biological Sciences Building
Indian Institute of Science
C.V. Raman Road
Bangalore - 560012
Karnataka
India

sathees@iisc.ac.in

+918022932674

Research inProf. Sathees C. Raghavan’s Laboratory