Jakub Onhajzer

The acquired JAK2 -V617F mutation plays a causal role in myeloproliferative neoplasms (MPN). Weakly activating JAK2 germline variants have been associated with MPN risk, but the underlying mechanisms remain unclear. We previously identified the JAK2 -R1063H germline variant, which contributes to hereditary MPN and increased disease severity in essential thrombocythemia. Here, we studied alterations in hematopoiesis in Jak2 -R1063H knock-in mice. The Jak2 -R1063H mouse cohort exhibited increased mortality, stimulated thrombopoiesis and elevated D-dimers levels, indicative of thrombotic complications. Bone marrow analysis revealed myeloid bias, enhanced megakaryopoiesis and activation of inflammatory signaling. Transcriptional and functional assays of hematopoietic stem cells suggested their accelerated aging and functional decline. The Egr1 transcriptional network, including the Thbs1 gene, progressively increased in aging mice, reinforcing alterations initiated by Jak2/Stat signaling. In murine acute myelogenous leukemia models, the Jak2 -R1063H cooperated with a driver oncogene in promoting leukemogenesis. Germline JAK2 -R1063H was found in 10 of 200 MPN patients from local hematology centers, with a higher minor allele frequency compared to healthy controls. Patients harboring JAK2 -R1063H variant exhibited an increased incidence of thrombotic complications and disease progression with shortened survival. In conclusion, our findings identify the JAK2 -R1063H germline variant as a risk factor for MPN development, thrombotic complications, and leukemic transformation.

Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant Kras, selective isolation and analysis of transforming or tumor cells that produce the Kras oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant Kras ^G12D that enables the “activation” of Kras ^G12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated Kras ^RR ) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the Kras ^RR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (Apc). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that Kras ^G12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the Kras ^G12D function is effectively suppressed by specific inhibitor MRTX1133.

The single-layer epithelium of the gastrointestinal tract is a dynamically renewing tissue that ensures nutrient absorption, secretory and barrier functions and is involved in immune responses. The basis for this homeostatic renewal is the Wnt signaling pathway. Blocking this pathway can lead to epithelial damage, while its abnormal activation can result in the development of intestinal tumors. In this study, we investigated the dynamics of intestinal epithelial cells and tumorigenesis using a conditional mouse model. Using single-cell and bulk RNA sequencing and histological analysis, we elucidated the cellular responses following the loss of specific cell types. We focused on the fate of cells in the lower parts of the intestinal crypts and the development of colon adenomas. By partially inactivating the transcription factor Tcf4, a key effector of the Wnt signaling pathway, we analyzed the regeneration of isolated hyperproliferative foci (crypts). Our results suggest that the damaged epithelium is not restored by a specific regeneration program associated with oncofetal gene production, but rather by a standard homeostatic renewal pathway. Moreover, disruption of Tcf4 in secretory progenitors resulted in a significant shift in the cell lineage from Paneth cells to goblet cells, characterized by morphological changes and loss of Paneth cell-specific genes. We also found that hyperactivation of the Wnt signaling pathway in colonic adenomas correlated with the upregulation of genes typical of Paneth cells in the intestine, followed by the emergence of secretory tumor cells producing the Wnt3 ligand. The absence of Tcf4 led to a phenotypic shift of the tumor cells towards goblet cells. Our study presents a new model of epithelial regeneration based on the genetically driven partial elimination of intestinal crypts. We highlight the critical role of Tcf4 in the control of cell lineage decisions in the intestinal epithelium and colon tumors.

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with a poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions, and its expression is maintained in most colorectal cancers (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli (Apc) tumor-suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with the transcription of genes involved in epithelial–mesenchymal transition, the regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by β-catenin and additionally by the Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/β-catenin signaling, YAP, and TROP2 expression.

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions and its expression is maintained in most colorectal cancer (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli ( Apc ) tumor suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with transcription of genes involved in epithelial-mesenchymal transition, regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by β-catenin and additionally by Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/β-catenin signaling, YAP, and TROP2 expression. Simple Summary: Colorectal cancer (CRC) is one of the most common cancers worldwide. While systemic treatment of CRC is based on chemotherapy, subsequent therapeutic options are far less effective. Trophoblast cell surface antigen 2 (TROP2) is highly expressed in many carcinomas, including CRC, where its expression correlates with poor prognosis. Anti-TROP2-targeted therapy was approved for the treatment of breast and urothelial carcinomas. We aimed to determine whether TROP2 is a suitable target for the treatment of CRC. We demonstrated that TROP2 expression in CRC correlates with lymph node metastasis and poor tumor differentiation. Analysis of mouse tumor models, patient-derived organoids, and tumor cells revealed that TROP2 expression is associated with features related to epithelial-mesenchymal transition and invasiveness. Our results suggest that TROP2 targeting may be a promising approach, especially in the early phase of treatment.