Silvia Schäfer

Abstract Mutational inactivation of VHL is the earliest genetic event in the majority of clear cell renal cell carcinomas (ccRCC), leading to accumulation of the HIF-1α and HIF-2α transcription factors. While correlative studies of human ccRCC and functional studies using human ccRCC cell lines have implicated HIF-1α as an inhibitor and HIF-2α as a promoter of aggressive tumour behaviours, their roles in tumour onset have not been functionally addressed. Herein we show using an autochthonous ccRCC model that Hif1a is essential for tumour formation whereas Hif2a deletion has only minor effects on tumour initiation and growth. Both HIF-1α and HIF-2α are required for the clear cell phenotype. Transcriptomic and proteomic analyses reveal that HIF-1α regulates glycolysis while HIF-2α regulates genes associated with lipoprotein metabolism, ribosome biogenesis and E2F and MYC transcriptional activities. HIF-2α-deficient tumours are characterised by increased antigen presentation, interferon signalling and CD8 + T cell infiltration and activation. Single copy loss of HIF1A or high levels of HIF2A mRNA expression correlate with altered immune microenvironments in human ccRCC. These studies reveal an oncogenic role of HIF-1α in ccRCC initiation and suggest that alterations in the balance of HIF-1α and HIF-2α activities can affect different aspects of ccRCC biology and disease aggressiveness.

ABSTRACT: In 2022, the European LeukemiaNet (ELN) risk stratification for patients with acute myeloid leukemia (AML) has been updated. We aimed to validate the prognostic value of the 2022 ELN classification (ELN22) by evaluating 1570 patients with newly diagnosed AML (median age, 56 years) treated with cytarabine-based intensive chemotherapy regimens. Compared with 2017 ELN classification (ELN17), which allocated 595 (38%), 413 (26%), and 562 patients (36%) to the favorable-, intermediate-, and adverse-risk categories, ELN22 classified 575 (37%), 410 (26%), and 585 patients (37%) as favorable, intermediate, and adverse risk, respectively. Risk group allocation was revised in 340 patients (22%). Most patients were reclassified into the ELN22 intermediate- or ELN22 adverse-risk group. The allocation of patients according to the ELN22 risk categories resulted in a significantly distinct event-free survival (EFS), relapse-free survival, and overall survival (OS). Compared with ELN17, reallocation according to the ELN22 recommendations resulted in a significantly improved prognostic discrimination for OS (3-year area under the curve, 0.71 vs 0.67). In patients with ELN22 favorable-risk AML, co-occurring myelodysplasia-related (MR) gene mutations did not significantly affect outcomes. Within the ELN22 adverse-risk group, we observed marked survival differences across mutational groups (5-year OS rate of 21% and 3% in patients with MR gene mutations and TP53 mutations, respectively). In patients harboring MR gene mutations, EZH2-, STAG2-, and ZRSR2-mutated patients showed an intermediate-like OS. In patients with secondary AML and those who underwent allogeneic hematopoietic cell transplantation, EFS and OS significantly differed between ELN22 risk groups, whereas the prognostic abilities of ELN17 and ELN22 classifications were similar. In conclusion, ELN22 improves prognostic discrimination in a large cohort of intensively treated patients with AML. Given the heterogeneous outcome in patients with MR gene alterations, ranging between those of intermediate and adverse risk patients, we suggest re-evaluation of risk allocation in these patients.

Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.

Lysine acetyltransferase 2 A (KAT2A) plays a pivotal role in epigenetic gene regulation across various types of cancer. In colorectal cancer (CRC), increased KAT2A expression is associated with a more aggressive phenotype. Our study aims to elucidate the molecular underpinnings of KAT2A dependency in CRC and assess the consequences of KAT2A depletion. We conducted a comprehensive analysis by integrating CRISPR-Cas9 screening data with genomics, transcriptomics, and global acetylation patterns in CRC cell lines to pinpoint molecular markers indicative of KAT2A dependency. Additionally, we characterized the phenotypic effect of a CRISPR-interference-mediated KAT2A knockdown in CRC cell lines and patient-derived 3D spheroid cultures. Moreover, we assessed the effect of KAT2A depletion within a patient-derived xenograft mouse model in vivo. Our findings reveal that KAT2A dependency is closely associated with microsatellite stability, lower mutational burden, and increased molecular differentiation signatures in CRC, independent of the KAT2A expression levels. KAT2A-dependent CRC cells display higher gene expression levels and enriched H3K27ac marks at gene loci linked to enterocytic differentiation. Furthermore, loss of KAT2A leads to decreased cell growth and viability in vitro and in vivo, downregulation of proliferation- and stem cell-associated genes, and induction of differentiation markers. Altogether, our data show that a specific subset of CRCs with a more differentiated phenotype relies on KAT2A. For these CRC cases, KAT2A might represent a promising novel therapeutic target.

Relapse in acute myeloid leukemia (AML) is driven by resistant subclones that survive chemotherapy. It is assumed that these resilient leukemic cells can modify their proliferative behavior by entering a quiescent-like state, similar to healthy hematopoietic stem cells (HSCs). These dormant cells can evade the effects of cytostatic drugs that primarily target actively dividing cells. Although quiescence has been extensively studied in healthy hematopoiesis and various solid cancers, its role in AML has remained unexplored. In this study, we applied an HSC-derived quiescence-associated gene signature to an AML patient cohort and found it to be strongly correlated with poor prognosis and active TGF-β signaling. In vitro treatment with TGF-β1 induces a quiescence-like phenotype, resulting in a G0 shift and reduced sensitivity to cytarabine. To find potential therapeutic targets that prevent AML-associated quiescence and improve response to cytarabine, we conducted a comprehensive CRISPR interference (CRISPRi) screen combined with TGF-β1 stimulation. This approach identified TGFBR1 inhibitors, like vactosertib, as effective agents for preventing the G0 shift in AML cell models. However, pretreatment with vactosertib unexpectedly induced complete resistance to cytarabine. To elucidate the underlying mechanism, we performed a multi-faceted approach combining a second CRISPRi screen, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and in silico analysis. Our findings revealed that TGFBR1 inhibitors unintentionally target the nucleoside transporter SLC29A1 (ENT1), leading to reduced intracellular cytarabine levels. Importantly, we found that this drug interaction is not unique to TGFBR1 inhibitors, but extends to other clinically significant kinase inhibitors, such as the FLT3 inhibitor midostaurin. These findings may have important implications for optimizing combination therapies in AML treatment.