Learn about our current projects!
Learn about our current projects!
Embedded Files
Umesh is currently focused on DNA damage and repair pathways in cancer cells, which involves studying the functional role of DNA Polymerase theta (Polθ), a main enzyme involved in repairs of DNA double-strand breaks through Theta-mediated end joining (TMEJ) and confers resistance to genotoxic agents. OTK-positive leukemia cells (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1) accumulate high levels of reactive oxygen species-induced oxidative DNA damage, but DNA repair pathways such as altered nucleotide excision repair, mismatch repair, Homologous Recombination (HR), and/or Non-Homologous end joining (NHEJ), but not TMEJ, activities protect them from apoptosis. However, endogenous aldehydes represent another class of metabolically derived, highly active chemicals that cause DNA damage. Overall, we demonstrated that Polθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.
Polθ mediated TMEJ activity is required to protect the cancer cells. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We have identified that PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing; however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. Overall, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ. DNA replication is constantly challenged by DNA damage and other intrinsic and extrinsic stresses collectively termed replication stress. When replication forks encounter DNA damage, they can stall or collapse, generating DNA double-strand breaks (DSBs) that trigger a DNA damage response and, if not adequately repaired, can lead to cell death. Polθ play role not only in the timing of DNA replication, but also in restarting replication forks, in repairing DSBs caused by replication forks' collapse, and also in filling of ssDNA gaps on lagging strands. However, the mechanisms responsible for Polθ recruitment to stalled replication forks remained unknown. We identified that PSF3, a member of GINS replicative complex, recruits Polθ. Altogether, his work demonstrated that Polθ plays an essential role in protecting cancer cells by repairing DNA lesions triggered by intrinsic and extrinsic stresses. Hence, Polθ can be utilized as a synthetic lethal target to achieve a therapeutic effect.
Monika is currently working on is focused on the clonal response of acute myeloid leukemia and myeloproliferative neoplasm to the treatment with DNA damage inhibitors and/or standard treatment regimens. Monika is involved in the clinical trial aiming to help patients with myeloproliferative neoplasms. To survive, AML and MPN clones become highly dependent on DNA damage response (DDR) pathways, including DSB sensors (ATM, ATR) and repair mechanisms such as homologous recombination (RAD51 - mediated HR; RAD52-mediated TA - HR and SSA), non-homologous end - joining (DNA - PK), as well as microhomology - mediated end - joining (PARP1/Polθ - dependent MMEJ). We have previously devised a patient-tailored “clonal attack” strategy that integrates single-cell targeted DNA sequencing (sctDNA - seq) - profiling up to 1,394 variants across 54 leukemia - driver genes -with in vitro sensitivity testing to DDR inhibitors (DDRi). We have identified distinct clonal subpopulations with variable sensitivities to inhibitors of ATR, PARP, ATM, and RAD52 in multiple AML and MPN samples. In the new approach we are combining DDR inhibitors with standard treatment in order to initiate the clonal sensitivity achieving synergistic elimination of all clones in vitro. The clonal medicine paradigm shifts leukemia treatment toward precision targeting of DDR vulnerabilities unique to individual clones, offering a powerful means to overcome therapeutic resistance while sparing normal cells. Now we propose that the combination of standard treatment with DDR inhibitors will be able to eradicate the leukemic clones even more robustly. This approach also proposes broad applicability across cancers.
Monika is extremely passionate about her work, and since 2023, she has also been the head of Fels Single Cell Multiomics Facility at Temple University. The facility advances high-resolution single-cell analysis by providing state-of-the-art technologies, expert support, and end-to-end services.
Viet is currently focused on the stabilization of ZEB1, promoting inefficiency of Polθ inhibitors in leukemia cells within the bone marrow microenvironment. Tumor microenvironment has been an interesting research topic in anti-cancer therapies. In hematopoietic malignancies, bone marrow is considered the major tumor microenvironment of blood cancer cells. As originating from bone marrow, leukemia cells have been reported to exploit supportive features of bone marrow microenvironment (BMM) to survive, proliferate and escape from therapeutic interventions. In DNA damage response, our group has reported the activation of TGFβR-SMAD3 signaling induces the resistance to PARP inhibitors in myeloid leukemia cells within the BMM. Moreover, we have discovered Polθ, a key protein in theta-mediated end joining (TMEJ) pathway, is a very promising therapeutic target anti-leukemia medicine. Therefore, it is pivotal to investigate the efficiency of Polθ inhibitors (Polθi) in leukemia cells within the BMM.
We preliminarily found the BMM reduced the expression of Polθ and the activity of Polθ-mediated TMEJ, in comparison to peripheral blood microenvironment. Thus, it resulted in the inefficiency of Polθi in leukemia cells within the BMM. ZEB1 has been reported to transcriptionally repress Polθ expression, decreasing Polθ-mediated TMEJ efficiency in breast cancer. Meanwhile, hypoxia (1-2% oxygen) is a major component in BMM, and we observed HIF-1α stabilized ZEB1 in leukemia cells within the BMM. Although ZEB1 selective inhibitor has not been developed, class I HDAC inhibitor mocetinostat has been shown to reduce ZEB1 expression. Altogether, we postulate the BMM promotes inefficiency of Polθi in leukemia cells, via the stabilization of ZEB1. Therefore, inhibition of ZEB1 by mocetinostat overcomes the inefficiency of Polθi in leukemia cells within the BMM.
Sri is currently working on DYRK1A. DYRK1A is a serine/threonine kinase encoded by the DYRK1A gene on chromosome 21 (chr21) that has been implicated in several neurological conditions, including Down syndrome and Alzheimer’s disease. Children with Down syndrome have a significantly higher risk of developing both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Overexpression of DYRK1A due to chr21 amplifications is a hallmark of a cohort of acute myeloid leukemia (AML) and affects the activity of the DREAM complex of genes regulating DNA damage response. However, the DYRK1A's contribution to leukemia development is largely unknown.
In our study, we used the Set-2 cell line, a human megakaryoblastic cell line that expresses the highest levels of DYRK1A protein among leukemia cell lines. DYRK1A was CRISPR/Cas9 knocked out to generate an isogenic pair of Set-2 cell lines displaying high levels of DYRK1A (DYRK1A+) and no DYRK1A (DYRK1A-). Protein expression studies suggested that the proteins like DNA polymerase theta (Polθ) and 53BP1 (limits DNA end-resection) were upregulated, and CtIP (promotes DNA end-resection), BRCA1, BRCA2, and RAD51 were downregulated in DYRK1A+ cells compared to the knockout counterparts. In concordance, DYRK1A+ cells displayed limited DNA end-resection and preferred Polθ-mediated microhomology-dependent (TMEJ) pathway to repair DNA double strand breaks (DSBs). Conversely, DYRK1A- cells displayed prolonged DNA end-resection and favored BRCA1/2 and RAD51-mediated homologous recombination (HR) pathway. Remarkably, DYRK1A+ cells were sensitive to the Polθ inhibitor and resistant to the RAD51 inhibitor when compared to DYRK1A- cells. Together, these results suggest that overexpression of DYRK1A promotes the TMEJ pathway in a cohort of chr21-amplified AML, and that Polθ-mediated TMEJ is a legitimate new therapeutic target.
Rifat's current research focus is to find out novel targeted therapy for P53-associated Acute Myeloid Lukemia (AML) which is dared to treat till date. Mutation or aberrant expression of the tumor suppressor gene TP53 turned the P53-associated AML is the most aggressive, extremely poor prognostic, often refractory to chemotherapy, and have a high rate of relapse even after allogeneic hematopoietic stem cell transplantation. So, it is utmost importance to find out efficient targeted therapy for this type of AML.
She is trying to discover new drugs to treat and eliminate the P53 mutant leukemias. By CRISPR mediated synthetic lethality screening, we successfully identified some exclusive targets of DNA Damage Repair (DDR) proteins and small molecule inhibitor of this targets would efficiently use to treat P53-associated AML. After screening the targets in AML cell lines, AML patient’s primary cells, she is now validating the efficacy of these small molecule inhibitors in P53 dependent humanized AML mouse model.
Sadia is currently working alongside Monika. She is assisting in her project pertaining to the clonal attack and DNA Damage Inhibitors (DDRis) with a combination of standard treatment. She is assisting in determining how the leukemic cells in vitro are reacting to the combination therapy and whether or not the cancer cells are dying due to the treatment. She hopes to create substantial datasets that would later be used in vivo.
Page updated
Report abuse