IU Indianapolis ScholarWorks :: Browsing by Author "Palam, Lakshmi Reddy"

Browsing by Author "Palam, Lakshmi Reddy"

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Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation
(Elsevier, 2019-02-12) Garg, Garima; Muschaweckh, Andreas; Moreno, Helena; Vasanthakumar, Ajithkumar; Floess, Stefan; Lepennetier, Gildas; Oellinger, Rupert; Zhan, Yifan; Regen, Tommy; Hiltensperger, Michael; Peter, Christian; Aly, Lilian; Knier, Benjamin; Palam, Lakshmi Reddy; Kapur, Reuben; Kaplan, Mark H.; Waisman, Ari; Rad, Roland; Schotta, Gunnar; Huehn, Jochen; Kallies, Axel; Korn, Thomas; Pediatrics, School of Medicine
Foxp3+ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and "toxic" gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.
Driver Mutations in Leukemia Promote Disease Pathogenesis through a Combination of Cell-Autonomous and Niche Modulation
(Elsevier, 2020-07-14) Ramdas, Baskar; Mali, Raghuveer Singh; Palam, Lakshmi Reddy; Pandey, Ruchi; Cai, Zhigang; Pasupuleti, Santhosh Kumar; Burns, Sarah S.; Kapur, Reuben; Pediatrics, School of Medicine
Studies of patients with acute myeloid leukemia (AML) have led to the identification of mutations that affect different cellular pathways. Some of these have been classified as preleukemic, and a stepwise evolution program whereby cells acquire additional mutations has been proposed in the development of AML. How the timing of acquisition of these mutations and their impact on transformation and the bone marrow (BM) microenvironment occurs has only recently begun to be investigated. We show that constitutive and early loss of the epigenetic regulator, TET2, when combined with constitutive activation of FLT3, results in transformation of chronic myelomonocytic leukemia-like or myeloproliferative neoplasm-like phenotype to AML, which is more pronounced in double-mutant mice relative to mice carrying mutations in single genes. Furthermore, we show that in preleukemic and leukemic mice there are alterations in the BM niche and secreted cytokines, which creates a permissive environment for the growth of mutation-bearing cells relative to normal cells.
Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells
(Elsevier, 2022) Ramdas, Baskar; Yuen, Lisa Deng; Palam, Lakshmi Reddy; Patel, Roshini; Pasupuleti, Santhosh Kumar; Jideonwo, Victoria; Zhang, Ji; Maguire, Callista; Wong, Eric; Kanumuri, Rahul; Zhang, Chujing; Sandusky, George; Chan, Rebecca J.; Zhang, Chi; Stieglitz, Elliot; Haneline, Laura; Kapur, Reuben; Pediatrics, School of Medicine
Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton’s tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.
Inhibition of Inflammatory Signaling in Tet2 Mutant Preleukemic Cells Mitigates Stress-Induced Abnormalities and Clonal Hematopoiesis
(Elsevier, 2018-12-06) Cai, Zhigang; Kotzin, Jonathan J.; Ramdas, Baskar; Chen, Sisi; Nelanuthala, Sai; Palam, Lakshmi Reddy; Pandey, Ruchi; Mali, Raghuveer Singh; Liu, Yan; Kelley, Mark R.; Sandusky, George; Mohseni, Morvarid; Williams, Adam; Henao-Mejia, Jorge; Kapur, Reuben; Pediatrics, School of Medicine
Inflammation is a risk factor for cancer development. Individuals with preleukemic TET2 mutations manifest clonal hematopoiesis and are at a higher risk of developing leukemia. How inflammatory signals influence the survival of preleukemic hematopoietic stem and progenitor cells (HSPCs) is unclear. We show a rapid increase in the frequency and absolute number of Tet2-KO mature myeloid cells and HSPCs in response to inflammatory stress, which results in enhanced production of inflammatory cytokines, including interleukin-6 (IL-6), and resistance to apoptosis. IL-6 induces hyperactivation of the Shp2-Stat3 signaling axis, resulting in increased expression of a novel anti-apoptotic long non-coding RNA (lncRNAs), Morrbid, in Tet2-KO myeloid cells and HSPCs. Expression of activated Shp2 in HSPCs phenocopies Tet2 loss with regard to hyperactivation of Stat3 and Morrbid. In vivo, pharmacologic inhibition of Shp2 or Stat3 or genetic loss of Morrbid in Tet2 mutant mice rescues inflammatory-stress-induced abnormalities in HSPCs and mature myeloid cells, including clonal hematopoiesis.
Loss of Dnmt3a impairs hematopoietic homeostasis and myeloid cell skewing via the PI3Kinase pathway
(The American Society for Clinical Investigation, 2023-05-08) Palam, Lakshmi Reddy; Ramdas, Baskar; Pickerell, Katelyn; Pasupuleti, Santhosh Kumar; Kanumuri, Rahul; Cesarano, Annamaria; Szymanski, Megan; Selman, Bryce; Dave, Utpal P.; Sandusky, George; Perna, Fabiana; Paczesny, Sophie; Kapur, Reuben; Pediatrics, School of Medicine
Loss-of-function mutations in the DNA methyltransferase 3A (DNMT3A) are seen in a large number of patients with acute myeloid leukemia (AML) with normal cytogenetics and are frequently associated with poor prognosis. DNMT3A mutations are an early preleukemic event, which - when combined with other genetic lesions - result in full-blown leukemia. Here, we show that loss of Dnmt3a in hematopoietic stem and progenitor cells (HSC/Ps) results in myeloproliferation, which is associated with hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway. PI3Kα/β or the PI3Kα/δ inhibitor treatment partially corrects myeloproliferation, although the partial rescue is more efficient in response to the PI3Kα/β inhibitor treatment. In vivo RNA-Seq analysis on drug-treated Dnmt3a-/- HSC/Ps showed a reduction in the expression of genes associated with chemokines, inflammation, cell attachment, and extracellular matrix compared with controls. Remarkably, drug-treated leukemic mice showed a reversal in the enhanced fetal liver HSC-like gene signature observed in vehicle-treated Dnmt3a-/- LSK cells as well as a reduction in the expression of genes involved in regulating actin cytoskeleton-based functions, including the RHO/RAC GTPases. In a human PDX model bearing DNMT3A mutant AML, PI3Kα/β inhibitor treatment prolonged their survival and rescued the leukemic burden. Our results identify a potentially new target for treating DNMT3A mutation-driven myeloid malignancies.
Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway
(American Society for Clinical Investigation, 2018-02-22) Palam, Lakshmi Reddy; Mali, Raghuveer Singh; Ramdas, Baskar; Srivatsan, Sridhar Nonavinkere; Visconte, Valeria; Tiu, Ramon V.; Vanhaesebroeck, Bart; Roers, Axel; Gerbaulet, Alexander; Xu, Mingjiang; Janga, Sarath Chandra; Takemoto, Clifford M.; Paczesny, Sophie; Kapur, Reuben; Pediatrics, School of Medicine
Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2-/- cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage-derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non-mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.
Obesity-induced inflammation exacerbates clonal hematopoiesis
(The American Society for Clinical Investigation, 2023-06-01) Pasupuleti, Santhosh Kumar; Ramdas, Baskar; Burns, Sarah S.; Palam, Lakshmi Reddy; Kanumuri, Rahul; Kumar, Ramesh; Pandhiri, Taruni Reddy; Dave, Utpal P.; Yellapu, Nanda Kumar; Zhou, Xinyu; Zhang, Chi; Sandusky, George E.; Yu, Zhi; Honigberg, Michael C.; Bick, Alexander G.; Griffin, Gabriel K.; Niroula, Abhishek; Ebert, Benjamin L.; Paczesny, Sophie; Natarajan, Pradeep; Kapur, Reuben; Medicine, School of Medicine
Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis of indeterminate potential (CHIP) is frequent in aging and involves the expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, the risk factors that contribute to CHIP-associated clonal hematopoiesis (CH) are poorly understood. Obesity induces a proinflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data for 47,466 individuals with validated CHIP in the UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in the waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1, and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and that a proinflammatory state could potentiate the progression of CHIP to more significant hematologic neoplasia. The calcium channel blockers nifedipine and SKF-96365, either alone or in combination with metformin, MCC950, or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP-mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in individuals with obesity.
Physioxia-induced downregulation of Tet2 in hematopoietic stem cells contributes to enhanced self-renewal
(American Society of Hematology, 2022) Aljoufi, Arafat; Zhang, Chi; Ropa, James; Chang, Wennan; Palam, Lakshmi Reddy; Cooper, Scott; Ramdas, Baskar; Capitano, Maegan L.; Broxmeyer, Hal E.; Kapur, Reuben; Microbiology and Immunology, School of Medicine
Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in differentiation when exposed to ambient air as opposed to physioxia. Mechanism(s) behind this distinction are poorly understood but have the potential to improve stem cell transplantation. Single-cell RNA sequencing of HSCs in physioxia revealed upregulation of HSC self-renewal genes and downregulation of genes involved in inflammatory pathways and HSC differentiation. HSCs under physioxia also exhibited downregulation of the epigenetic modifier Tet2. Tet2 is α-ketoglutarate, iron- and oxygen-dependent dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine, thereby promoting active transcription. We evaluated whether loss of Tet2 affects the number and function of HSCs and hematopoietic progenitor cells (HPCs) under physioxia and ambient air. In contrast to wild-type HSCs (WT HSCs), a complete nonresponsiveness of Tet2-/- HSCs and HPCs to changes in oxygen tension was observed. Unlike WT HSCs, Tet2-/- HSCs and HPCs exhibited similar numbers and function in either physioxia or ambient air. The lack of response to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in self-renewal and quiescence genes among WT HSC-physioxia, Tet2-/- HSC-physioxia and Tet2-/- HSC-air. We define a novel molecular program involving Tet2 in regulating HSCs under physioxia.
Potential clinical use of azacitidine and MEK inhibitor combination therapy in PTPN11-mutated juvenile myelomonocytic leukemia
(Elsevier, 2023) Pasupuleti, Santhosh Kumar; Chao, Karen; Ramdas, Baskar; Kanumuri, Rahul; Palam, Lakshmi Reddy; Liu, Sheng; Wan, Jun; Annesley, Colleen; Loh, Mignon L.; Stieglitz, Elliot; Burke, Michael J.; Kapur, Reuben; Pediatrics, School of Medicine
Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative neoplasm of childhood. The molecular hallmark of JMML is hyperactivation of the Ras/MAPK pathway with the most common cause being mutations in the gene PTPN11, encoding the protein tyrosine phosphatase SHP2. Current strategies for treating JMML include using the hypomethylating agent, 5-azacitidine (5-Aza) or MEK inhibitors trametinib and PD0325901 (PD-901), but none of these are curative as monotherapy. Utilizing an Shp2E76K/+ murine model of JMML, we show that the combination of 5-Aza and PD-901 modulates several hematologic abnormalities often seen in JMML patients, in part by reducing the burden of leukemic hematopoietic stem and progenitor cells (HSC/Ps). The reduced JMML features in drug-treated mice were associated with a decrease in p-MEK and p-ERK levels in Shp2E76K/+ mice treated with the combination of 5-Aza and PD-901. RNA-sequencing analysis revealed a reduction in several RAS and MAPK signaling-related genes. Additionally, a decrease in the expression of genes associated with inflammation and myeloid leukemia was also observed in Shp2E76K/+ mice treated with the combination of the two drugs. Finally, we report two patients with JMML and PTPN11 mutations treated with 5-Aza, trametinib, and chemotherapy who experienced a clinical response because of the combination treatment.
REGULATION OF CHOP TRANSLATION IN RESPONSE TO eIF2 PHOSPHORYLATION AND ITS ROLE IN CELL FATE
(2012-05) Palam, Lakshmi Reddy; Wek, Ronald C.; Herring, Brian P.; Harris, Robert A. (Robert Allison), 1939-; Skalnik, David G.
In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.

Browsing by Author "Palam, Lakshmi Reddy"

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