Biology subjects

Kubaczka, C.

Publications and source records attributed to Kubaczka, C..

6 recordsLinked to original sources

bioRxiv2026

RNA splicing factor mutations drive myeloid neoplasm oncogenesis through protein complex poisoning

RNA splicing factor mutations (SFmut) are founding oncogenic events which cause RNA splicing errors with unpredictable gene expression dynamics. Despite extensive transcriptomic studies, SFmut cancer-initiating mechanisms remain elusive. Among SFmut cancers, myeloid neoplasms (MN) alone offer a setting where true cancer-driving SFmut stem cells can be identified, namely the hematopoietic stem cell (HSC). Within a cohort of 62 MN patients and 20 healthy donors, we conducted long/short-read single-cell transcriptomics (10X-ONT, n = 21) and immunophenotype-resolved low-cell proteomics (pauciproteomics, n = 78) to resolve gene expression, RNA splicing and protein expression dynamics across healthy and SFmut MN hematopoiesis. During hematopoietic differentiation, SFmut RNA mis-splicing decouples proteotranscriptomic dynamics in a mutation-specific manner. Pauciproteomics defines the functional effects of SFmut RNA mis-splicing on the protein layer, identifying early-stage protein effects which poison entire functional protein complexes and progressively disrupt the proteome-wide network during cellular maturation. Importantly, this information could neither be resolved nor predicted by transcriptomic analyses. Finally, we functionally validate the proteome dynamics of HSC through induced pluripotent stem cell culture models of early hematopoietic differentiation, identifying candidate mechanisms for SFmut oncogenesis. Overall, these data elucidate SFmut MN disease biology with unprecedented granularity, defining the molecular consequences of SFmut RNA mis-splicing and offering a generalizable framework for cancer stem cell studies.

cancer biology↗

bioRxiv2026

Flow-Induced Yap/Taz Signaling Balances Endothelial and Hematopoietic Stem Cell Fates

Mechanical forces from blood flow are essential for production of hematopoietic stem and progenitor cells (HSPCs) during embryogenesis, but the molecular mechanisms by which hemodynamic cues are sensed and orchestrate endothelial-to-hematopoietic (EHT) transition remain incompletely defined. We previously identified YAP mechanotransduction as a key integrator of physical forces with EHT. Here we show that hemodynamic forces can activate YAP signaling via the mechanoresponsive ion channel Piezo1 in human iPSC-derived hemogenic endothelium (HE) and zebrafish embryos. Investigation of the Piezo1/YAP axis revealed shared and unique roles of YAP and its paralogue TAZ in EHT. Mechanistically, we find a requirement for the Tead DNA-binding co-factor in YAP/TAZ-dependent control of HSPC number, and note that TAZ uniquely augments transcriptional output of the hematopoietic master regulator Runx1 via direct protein-protein interactions. By comprehensive scRNA-sequencing of YAP/TAZ gain-of-function (GOF) and yap-deficient cells from zebrafish, we reveal that YAP/TAZ promotes HSC production by positively regulating gene programs for hematopoietic self-renewal, cell cycle, and glycolysis-to-oxidative phosphorylation switching, while preventing reversion to endothelial identity. Importantly, comparison of GOF transcriptomes and functional analyses suggest decoupling of metabolic/proliferative and endothelial gene regulatory modules between YAP and TAZ: while either can functionally compensate for loss of the other in EHT, indiscriminate overactivation of TAZ enhances an endothelial program over pro-hematopoietic fate, ultimately blunting progression of HSPC production. Given that hemodynamic cues are integrated simultaneously by arterial and HE cells in embryonic vessels in which EHT occurs, these findings have strong implications for strategies designed to introduce biomechanical cues to in vitro hematopoietic differentiation systems to drive HSC production.

Cell Biology↗

bioRxiv2026

Efficient Generation of Functional TCRαβ+ Cytotoxic T Cells from hiPSCs via Small-Molecule Modulation

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3TCR{beta} cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4 immature single-positive (ISP) cells, markedly increasing CD4CD8 populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34 cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

cell biology↗

bioRxiv2025

RNA Editors Sculpt the Transcriptome During Terminal Erythropoiesis

Selective RNA degradation during terminal erythropoiesis results in a globin-rich transcriptome in mature erythrocytes, but the specific RNA decay pathways remain unknown. We found that deficiency of the terminal uridylyl transferase enzyme Zcchc6 and the 3-5 exoribonuclease Dis3l2 in mouse models led to fetal and perinatal reticulocytosis, an accumulation of RNA-rich precursors of terminal erythroid cells, suggesting their crucial roles in terminal red cell differentiation. Notably, knockout embryos exhibited persistent high-level expression of Hbb-bh1 globin, the ortholog of human fetal{gamma} -globin. Perturbation of the Zcchc6-Dis3l2 pathway in mice engineered to express the human {beta}-globin locus likewise increased{gamma} -globin levels in fetal erythroid cells, suggesting that globin switching entails post-transcriptional mechanisms of mRNA destabilization in addition to transcriptional down-regulation. We cultured human hematopoietic stem and progenitor cells (HSPCs), performed CRISPR/Cas9-mediated knockout of ZCCHC6 and DIS3L2, and observed accumulation of RNA and elevated {gamma}-globin levels in terminal erythroid cells. Our findings reveal a conserved role for the ZCCHC6/DIS3L2 RNA editors in terminal erythropoiesis and demonstrate a post-transcriptional mechanism for{gamma} -globin gene switching, advancing research into in vitro erythrocyte generation and{gamma} -globin stabilization to ameliorate hemoglobinopathies.

developmental biology↗

bioRxiv2024

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFy, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFy. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo, revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

cell biology↗

bioRxiv2021

Induction of peri-implantation stage synthetic embryos using reprogramming paradigms in ESCs

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that synthetic embryo-like structures are generated solely based on transcription factor-mediated molecular reprogramming of embryonic stem cells in a simple 3D co-culture system. ESCs in these cultures self-organize into elongated, compartmentalized synthetic embryo-like structures over the course of reprogramming exhibiting anterior visceral endoderm formation and symmetry breaking. Single-cell RNA-Seq reveals transcriptional profiles resembling epiblast, visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5-E5.5. Within the epiblast, compartment marker gene expression supports primordial germ cell specification. After transplantation, synthetic embryo-like structures implant in uteri and initiate the formation of decidual tissues. This system allows for fast and reproducible generation of synthetic embryo-like structures, providing further insights into synthetic embryology.

synthetic biology↗