Biology subjects

Krishnamurthy, M.

Publications and source records attributed to Krishnamurthy, M..

11 recordsLinked to original sources

bioRxiv2025

Human pancreatic organoids derived from pluripotent stem cells recapitulate pancreatic organogenesis

Pancreas organogenesis relies on sequential interactions between the pancreatic epithelium and surrounding mesodermal cell types that initiate epithelial budding and branching to form a complex ductal network with terminal acini. Despite recent advances with pluripotent stem cell-based approaches, there are no models that robustly recapitulate pancreas morphogenesis or the spatial organization of ductal, exocrine, endocrine and mesenchymal cells seen in the native organ. Here, we introduce a new pluripotent stem cell-based pancreatic organoid that captures the complexity seen during pancreatic development, with budding and stratification of multipotent progenitors followed by formation of a ductal network that give rise to peripheral acini. We identify a critical role for mesenchyme-derived factors to robustly promote pancreatic organoid formation and morphogenesis. Human pancreatic organoids are correctly patterned, with functional exocrine acini secreting digestive enzymes into a ductal network. Comparative analysis confirms that the pancreatic organoids are similar to early second trimester human pancreas, with potential to further mature upon transplantation in mice. Finally, we show that endocrinogenesis can be reproduced in organoids, generating functional islet-like clusters interspersed within the ductal network. Together, this represents an exciting new platform to study human pancreas development and a broad array of pancreatic diseases.

developmental biology↗

bioRxiv2025

Lamin A/C Deficiency Drives Genomic Instability and Poor Survival in Small-Cell Lung Cancer through Increased R-loop Accumulation

Lamin A/C (LMNA), a key component of the nuclear envelope, is essential for maintaining nuclear integrity and genome organization [1]. While LMNA dysregulation has been implicated in genomic instability across cancer and aging, the underlying mechanisms remain poorly understood [2]. Here, we investigate LMNAs role in small-cell lung cancer (SCLC), a highly aggressive malignancy characterized by extreme genomic instability [3, 4]. We demonstrate that LMNA depletion promotes R-loop accumulation, transcription-replication conflicts, replication stress, DNA breaks, and micronuclei formation. Mechanistically, LMNA loss disrupts nuclear pore complex distribution, reducing phenylalanine-glycine (FG)-nucleoporin incorporation and impairing RNA export efficiency. Furthermore, we show that LMNA expression is epigenetically repressed by EZH2 during SCLC differentiation from neuroendocrine (NE) to non-NE states. Clinically, low LMNA levels correlate with significantly worse survival in SCLC patients. These findings uncover a novel role for LMNA in safeguarding genome integrity and shaping tumor heterogeneity, with broad implications for cancer and aging. Significance StatementLamin A/C, a key structural component of the nuclear envelope, is frequently lost or mutated in cancer, laminopathies, and aging-related disorders. Lamin A/C loss is associated with genomic instability, but the underlying mechanisms remain incompletely understood. We demonstrate that LMNA loss drives genomic instability by promoting R-loop accumulation through disrupted nuclear pore dynamics and impaired RNA export. These findings reveal a previously unrecognized link between LMNA loss, nuclear envelope dysfunction, and genome instability. Targeting this pathway could help mitigate genomic instability in aging and laminopathies, while leveraging R-loop accumulation may enhance the efficacy of DNA-damaging therapies in cancer.

cancer biology↗

bioRxiv2024

Metastatic organotropism in small cell lung cancer

Metastasis is the leading cause of cancer-related deaths, yet its regulatory mechanisms are not fully understood. Small-cell lung cancer (SCLC) is the most metastatic form of lung cancer, with most patients presenting with widespread disease, making it an ideal model for studying metastasis. However, the lack of suitable preclinical models has limited such studies. We utilized rapid autopsy-derived tumors to develop xenograft models that mimic key features of SCLC, including histopathology, rapid and widespread development of metastasis to the liver, brain, adrenal, bone marrow, and kidneys within weeks, and response to chemotherapy. By integrating in vivo lineage selection with comprehensive bulk and single cell multiomic profiling of transcriptomes and chromatin accessibility, we identified critical cellular programs driving metastatic organotropism to the liver and brain, the most common sites of SCLC metastasis. Our findings reveal the key role of nuclear-cytoskeletal interactions in SCLC liver metastasis. Specifically, the loss of the nuclear envelope protein lamin A/C, encoded by the LMNA gene, increased nuclear deformability and significantly increased the incidence of liver metastasis. Human liver metastases exhibited reduced LMNA expression compared to other metastatic sites, correlating with poorer patient outcomes and increased mortality. This study introduces novel preclinical models for SCLC metastasis and highlights pathways critical for organ-specific metastasis, offering new avenues for the development of targeted therapies to prevent or treat metastatic disease.

cancer biology↗

bioRxiv2024

A non-tethering role for the Drosophila Pol θ linker domain in promoting damage resolution

DNA polymerase theta (Pol {theta}) is an error-prone translesion polymerase that becomes crucial for DNA double-strand break repair when cells are deficient in homologous recombination or non-homologous end joining. In some organisms, Pol {theta} also promotes tolerance of DNA interstrand crosslinks. Due to its importance in DNA damage tolerance, Pol {theta} is an emerging target for treatment of cancer and disease. Prior work has characterized the functions of the Pol {theta} helicase-like and polymerase domains, but the roles of the linker domain are largely unknown. Here, we show that the Drosophila melanogaster Pol {theta} linker domain promotes egg development and is required for tolerance of DNA double-strand breaks and interstrand crosslinks. While a linker domain with scrambled amino acid residues is sufficient for DNA repair, replacement of the linker with part of the Homo sapiens Pol {theta} linker or a disordered region from the FUS RNA-binding protein does not restore function. These results demonstrate that the linker domain is not simply a random tether between the helicase-like and polymerase domains. Furthermore, they suggest that intrinsic amino acid residue properties, rather than protein interaction motifs, are more critical for Pol {theta} linker functions in DNA repair.

molecular biology↗

bioRxiv2024

Microenvironment Shapes Cell State, Plasticity, and Heterogeneity of Small Cell Lung Cancer

Small-cell lung cancer (SCLC) is the most fatal form of lung cancer. Intra-tumoral heterogeneity, marked by neuroendocrine (NE) and non-neuroendocrine (non-NE) cell states, defines SCLC, but the drivers of SCLC plasticity are poorly understood. To map the landscape of SCLC tumor microenvironment (TME), we apply spatially resolved transcriptomics and quantitative mass spectrometry-based proteomics to metastatic SCLC tumors obtained via rapid autopsy. The phenotype and overall composition of non-malignant cells in the tumor microenvironment (TME) exhibits substantial variability, closely mirroring the tumor phenotype, suggesting TME-driven reprogramming of NE cell states. We identify cancer-associated fibroblasts (CAF) as a crucial element of SCLC TME heterogeneity, contributing to immune exclusion, and predicting exceptionally poor prognosis. Together, our work provides a comprehensive map of SCLC tumor and TME ecosystems, emphasizing their pivotal role in SCLCs adaptable nature, opening possibilities for re-programming the intercellular communications that shape SCLC tumor states.

cancer biology↗

bioRxiv2023

Human organoid modeling of congenital malformations caused by RFX6 mutations reveal an essential role for this transcription factor in establishing and maintaining duodenal identity upstream of PDX1

The gastrointestinal (GI) tract consists of highly specialized organs from the proximal esophagus to the distal colon, each with unique functions. Rare congenital malformations of the GI tract, including organ atresia, agenesis or mis-patterning are linked to gene mutations although the molecular basis of these malformations has been poorly studied due to lack of model systems to study human development. We identified a patient with compound heterozygous mutations in the transcription factor RFX6 with pancreatic agenesis as previously described. In addition, the patient had duodenal mal-rotation and atresia suggesting that establishment of the proximal small intestine was impaired in these patients. To identify the molecular basis of the intestinal malformation we generated induced pluripotent stem cell lines from this patient, and derived human intestinal organoid (HIOs) to identify how mutations in RFX6 impact intestinal patterning and function. We identified that the duodenal identity of HIOs and patient tissues had adopted a more distal small intestinal signature, including expression of SATB2, normally expressed in the ileum and colon. CRISPR-mediated correction of RFX6 restored duodenal identity, including expression of PDX1, which is required for duodenal development. Using transcriptomic approaches in HIOs and Xenopus embryos we identified that PDX1 is a downstream transcriptional target of RFX6 and that PDX1 expression in a RFX6 mutant background was sufficient to rescue duodenal identity. However, RFX6 had a PDX1-independent role in regulating expression of components of WNT, HH, and BMP signaling pathways that are critical for establishing early regional identity in the GI tract. In summary, we have identified that RFX6 is one of the most upstream regulators early intestinal patterning in vertebrates and that it acts by regulating key transcriptional and signaling pathways.

developmental biology↗

bioRxiv2023

Phenomenological modeling reveals the emergent simplicity of host-associated microbiomes

A key step towards rational microbiome engineering is in silico sampling of realistic microbial communities that correspond to desired host phenotypes, and vice versa. This remains challenging due to a lack of generative models that simultaneously capture compositions of host-associated microbiomes and host phenotypes. To that end, we present a generative model based on the mechanistic consumer/resource (C/R) framework. In the model, variation in microbial ecosystem composition arises due to differences in the availability of effective resources (inferred latent variables) while species resource preferences remain conserved. The same latent variables are used to model phenotypic states of hosts. In silico microbiomes generated by our model accurately reproduce universal and dataset-specific statistics of bacterial communities. The model allows us to address three salient questions in host-associated microbial ecologies: (1) which host phenotypes maximally constrain the composition of the host-associated microbiomes? (2) how context-specific are phenotype/microbiome associations, and (3) what are plausible microbiome compositions that correspond to desired host phenotypes? Our approach aids the analysis and design of microbial communities associated with host phenotypes of interest.

systems biology↗

bioRxiv2022

SMbiot: A Shared Latent Model for Microbiomes and their Hosts

The collective nature of the variation in host associated microbial communities suggest that they exhibit low dimensional characteristics. To identify these lower dimensional descriptors, we propose SMbiot (pronounced SIM BY OT): a Shared Latent Model for Microbiomes and their hosts. In SMbiot, latent variables embed host-specific microbial communities in a lower dimensional space and the corresponding features reflect controlling axes that dictate community compositions. Using data from different animal hosts, organ sites, and microbial kingdoms of life, we show that SMbiot identifies a small number of host-specific latent variables that accurately capture the compositional variation in host associated microbial communities. By using the same latents to describe hosts phenotypic states and the host-associated microbiomes, we show that the latent space embedding is informed by host physiology as well as the associated microbiomes. Importantly, SMbiot enables the quantification of host phenotypic differences associated with altered microbial community compositions in a host-specific manner, underscoring the context specificity of host-microbiome associations. SMbiot can also predict missing host metadata or microbial community compositions. This way, SMbiot is a concise quantitative method to understand the low dimensional collective behavior of host-associated microbiomes.

systems biology↗

bioRxiv2022

Drosophila Rif1 is critical for repair following P-element excision and influences pathway choice at double-strand breaks

Rif1 plays important roles in the repair of DNA double-strand breaks in multiple organisms. In mammals, RIF1 promotes non-homologous end joining and suppresses homologous recombination by interacting with 53BP1 to inhibit resection. In Saccharomyces cerevisiae, Rif1 directly binds DNA to inhibit resection and promote non-homologous end-joining. Yeast Rif1 can also facilitate long-range resection and promote single-strand annealing. Since it is not clear if Rif1 regulates resection-mediated pathway choice in other eukaryotes, we explored the role of Rif1 in double-strand break repair in Drosophila melanogaster. We found that rif1 mutants are not sensitive to ionizing radiation or hydroxyurea, demonstrating that it is not essential for the resolution of DNA damage in Drosophila. However, we show that rif1 null mutants are largely unable to repair a specific type of double-strand break that is induced upon the excision of a P-element transposon. Furthermore, assessment of repair pathway choice at I-SceI-induced breaks revealed Rif1 suppresses homologous recombination and promotes single-strand annealing. Collectively, our findings illustrate Drosophila Rif1 shares functions with both its yeast and mammalian counterparts and serves a unique role in repairing P-element-induced double-strand breaks.

genetics↗

bioRxiv2021

Engineering functional human gastrointestinal organoid tissues using the three primary germ layers separately derived from pluripotent stem cells

The development of human organoid model systems has provided new avenues for patient-specific clinical care and disease modeling. However, all organoid systems are missing important cell types that, in the embryo, get incorporated into organ tissues during development. Based on the concept of how embryonic organs are assembled, we developed an organoid assembly approach starting with cells from the three primary germ layers; enteric neuroglial, mesenchymal, and epithelial precursors, all separately derived from human pluripotent stem cells. From these we generated human gastric tissue containing differentiated glands, surrounded by layers of smooth muscle containing functional enteric neurons that controlled contractions of the engineered tissue. We used this highly tractable system to identify essential roles for the enteric nervous system in the growth and regional identity of the gastric epithelium and mesenchyme and for glandular morphogenesis of the antral stomach. This approach of starting with separately-derived germ layer components was applied to building more complex fundic and esophageal tissue, suggesting this as a new paradigm for tissue engineering.

developmental biology↗

bioRxiv2021

Using Human Induced Pluripotent Stem Cell Derived Organoids to Identify New Pathologies in Patients with PDX1 Mutations

Two patients with mutations in PDX1 presented with pancreatic agenesis, chronic diarrhea and poor weight gain, the causes of which were not identified through routine clinical testing. We generated patient derived organoids as a novel diagnostic strategy and observed that PDX1188delC/188delC antral organoids convert to an intestinal phenotype, while intestinal organoids undergo gastric metaplasia with significant reduction in enteroendocrine cells. This prompted a re-examination of gastric and intestinal biopsies from both PDX1188delC/188delC patients, which recapitulated organoid phenotypes. Antral biopsies had increased parietal cells and lacked G-cells suggesting loss of antral identity. These patients will now be monitored for the progression of metaplasia. This study demonstrates the utility of organoids for patient diagnoses and treatment.

developmental biology↗