The repeatability of evolution is fundamentally important for understanding the origin and diversification of life as well as for developing evolutionary forecasting tools. Repeatability is limited by stochasticity, here defined as changes that are independent of genotypic fitness effects. Over short timescales, the two main sources of stochasticity of evolutionary change are environmental stochasticity and demographic (life-history) stochasticity. Quantifying the effect of these two sources of stochasticity and how they interact in driving fitness outcomes is crucially important for predicting contemporary evolutionary responses. To gain insights in the effects of stochasticity, five institutes replicated an evolutionary experiment exposing Caenorhabditis elegans to novel rearing conditions. Replication across the institutes led to variation in selective environments, including through divergent microbiomes among institutes. Replication within institutes was done across demographic treatments that influence the potential for population-size dependent fluctuations in allele frequencies (drift) and genetic hitchhiking (draft). We found high among-institute variation in fitness outcomes, which was partially explained by variation in microbiota. Whereas lab-specific effects explained most of the variance in mean fitness, the repeatability of fitness outcomes depended more on demographic heterogeneity. Specifically, population bottlenecks resulted in high among-replicate variation in fitness. When combined, environmental and demographic stochasticity additively reduced repeatability, underlining their additive importance in developing evolutionary forecasting tools. These results further highlight the importance of statistically integrating heterogeneity in experimental evolution to identify factors constraining outcome repeatability and study replicability.

Epigenetic modifications are dynamic and reversible, making them attractive targets for therapeutic intervention in cancer. Although several drugs targeting epigenetic modifications (epidrugs) have been clinically approved, their application in T-cell acute lymphoblastic leukemia (T-ALL) remains limited, and predictive biomarkers of response are lacking. Here, we present a mass spectrometry (MS)-based pharmacoepigenetic approach to profile histone post-translational modifications (hPTMs) to identify signatures associated with drug sensitivity in T-ALL . Baseline hPTM landscapes were previously established by our group for 21 T-ALL cell lines using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Here, we treated these cell lines with a panel of nine drugs including histone deacetylase inhibitors and DNA methyltransferase inhibitors (epidrugs), alongside anthracyclines, which were included due to their known chromatin-related effects. Correlation of cell viability data with hPTM levels revealed distinct hPTM signatures linked to sensitivity for each drug class. These signatures were subsequently evaluated in T-ALL patient-derived xenograft (PDX) models. However, our analysis revealed substantial discrepancies in hPTM sensitivity signatures compared to those observed in vitro. Co-variation network analysis highlighted divergence in hPTM-hPTM correlation between the two models, underscoring limitations of cell lines for modeling dynamic epigenetic regulation in vivo. Our findings establish a framework for MS-based hPTM profiling in T-ALL and emphasize the importance of model selection in developing predictive epigenetic biomarkers.

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous hematologic malignancy in which LMO2 γδ-like T-ALL represents a rare but clinically aggressive subtype associated with poor treatment response and inferior survival. Integrated transcriptomic analyses identified high SOX11 expression as a defining feature of high-risk LMO2 γδ-like T-ALL, where elevated SOX11 levels correlated with refractory disease and poor clinical outcome. To investigate the functional role of SOX11 in γδ T-cell biology and leukemogenesis, we generated a conditional R26-SOX11 mouse model enabling lineage-specific SOX11 overexpression in T-cell progenitors. SOX11 expression promoted expansion of the innate γδ T-cell compartment in thymus, spleen, and bone marrow, accompanied by transcriptional activation of γδ T-cell differentiation, activation, and cytotoxicity programs. However, SOX11 overexpression alone was insufficient to induce leukemia or confer thymocyte self-renewal capacity. In contrast, combined SOX11 and LMO2 overexpression markedly accelerated T-ALL development and strongly increased the incidence of γδ-like leukemias, thereby recapitulating the human high-risk LMO2 γδ-like T-ALL subtype. Mechanistically, SOX11 expanded the pre-leukemic DN3 thymocyte compartment in LMO2-driven mouse model while promoting differentiation toward the γδ lineage. Transcriptomic profiling identified activation of MYCN-associated transcriptional programs in SOX11/LMO2 pre-leukemic thymocytes. Consistently, MYCN was highly expressed in human LMO2 γδ-like T-ALL, and recurrent stabilizing MYCN P44L mutations were enriched in this subtype. Functional validation using genetic and transplantation-based mouse models demonstrated that SOX11 cooperates with MYCN to accelerate T-ALL onset. Together, these findings establish a cooperative SOX11–MYCN oncogenic axis driving γδ-like T-ALL and provide a novel preclinical model for investigating therapeutic vulnerabilities in this high-risk leukemia subtype.

Glucocorticoids (GC) are cornerstone drugs in the treatment of multiple myeloma (MM). Because MM cells exploit the bone marrow microenvironment to obtain growth and survival signals, resistance to glucocorticoid-induced apoptosis emerges, yet the underlying mechanisms remain poorly characterized. Here, we identify that the chemokine receptor CCR1, together with its main ligand CCL3, plays a pivotal role in reducing the glucocorticoid sensitivity of MM cells. We show that blocking CCR1 signaling with the antagonist BX471 enhances the anti-MM effects of the glucocorticoid dexamethasone in MM cell lines, primary patient material and a myeloma xenograft mouse model. Mechanistically, the drug combination shifts the balance between pro- and antiapoptotic proteins towards apoptosis and deregulates lysosomal proteins. Our findings suggest that CCR1 may play a role in glucocorticoid resistance, as the GC-induced downregulation of CCR1 mRNA and protein is blunted in a GC-resistance onset model. Moreover, we demonstrate that inhibiting CCR1 partially reverses this resistance, providing a promising strategy for resensitizing MM cells to GC treatment.

Background: Mantle cell lymphoma (MCL) is a highly aggressive B cell lymphoma that accounts for 6% of all non-Hodgkin lymphomas and is characterized by abnormal proliferation of mature antigen- naive B lymphocytes. Despite recent therapeutic advances, MCL remains incurable due to frequent relapses and resistance to therapy. SOX11 is a key transcription factor in the pathogenesis of MCL and is highly expressed in conventional MCL, but it is not expressed in the normal B cells or in the indolent leukemic nonnodal MCL subtype. Although SOX11 was identified as crucial oncogene that supports maintenance of MCL, its role in the initiation of MCL remains largely unknown. Aims: We want to investigate if SOX11 overexpression is a driver MCL-like lymphoma in mice. Furthermore, we want to identify the cell-of-origin of this murine MCL model and uncover the molecular mechanisms underlying SOX11-driven B2-to-B1a reprogramming and formation of MCL-like disease. Methods: We developed a novel mouse model for condition overexpression of SOX11 and an eGFP reporter. We used different Cre-lines to restrict SOX11 expression in space (pre-B cells or common lymphoid progenitors (CLPs)) and time (tamoxifen-dependent Cre). Transplantation experiments are used to identify the cell-of-origin and asses B-cell reprogramming. We used a multi-omics approach, including scRNAseq and ATACseq, to unravel SOX11-based transcriptional activity. Results: We found that elevated levels of SOX11 in B-cells are sufficient to drive MCL-like lymphomas in mice. Moreover, SOX11 expression synergized with loss of p53 and overexpression of Ccnd2 to form aggressive MCL-like CD19+CD5+CD23-IgM+IgD+ lymphomas, with a median survival of 314 (p53Mb1; n=11), 165 (SOX11/p53Mb1; n=19), and 134 (SOX11/Ccnd2/p53Mb1; n=15) days. These SOX11-driven lymphomas show transcriptional, immunophenotypic and functional similarities with both murine B1a cells and MCL patients. scRNAseq analysis shows that SOX11 overexpression from CLP stage blocks T-cell development and skews B-cell development towards the B1a and marginal zone B-cell (MZB) lineage. Strikingly, while SOX11-induced B1a cells were dependent on MALT1 protease activity, the SOX11-induced MZBs were not. In contrast to our cyclin D2-driven MCL-like model, SOX11is inducing a pre-lymphoma stage, in which there is a progressive accumulation of B1a cells in peripheral blood, peritoneal cavity, spleen and bone-marrow. The pre-lymphoma B1a cells have a BCR repertoire which was biased towards binding of self-antigens, such as phosphatidylcholine, and have increased BCR signaling activity. To investigate how elevated SOX11 levels contribute to the pre-lymphoma phase, we explored three potential mechanisms: (1) expansion of existing B1a cells, (2) a developmental bias towards B1a cells, (3) or reprogramming of B2-cells towards a B1a cell fate. To test the first two hypothesis, we transplanted embryonic-derived B cells from fetal liver or bone marrow-derived B2 progenitors with and without SOX11 overexpression into secondary recipients. We found that only SOX11-expressing cells were capable of initiating MCL-like lymphomas. To evaluate the reprogramming potential, we crossed SOX11/p53 mice with a tamoxifen-inducible CreERT2 line and transplanted different sorted B1 and B2 subsets in immunocompromised mice followed by tamoxifen administration via diet. These experiments demonstrate that SOX11 overexpression is sufficient to reprogram mature B2 cells into a B1a-like phenotype, which subsequently expands and serves as the primary source of B1a/MCL-like lymphomagenesis Summary/Conclusion SOX11 functions as a key regulator of the B1a lineage in mice, and its aberrant expression drives the reprogramming of B2 to B1a cells, leading to the development of MCL-like lymphomas.

Background: Under sufficient iodine supply, dual oxidase (DUOX)-dependent H2O2 production constitutes the limiting factor for thyroid hormone (TH) synthesis. Inherited loss-of-function mutations in related genes can trigger congenital hypothyroidism (CH). TH supplementation is not always well-tolerated and requires dose adjustments throughout life. Regenerative medicine directed at thyroid follicular cells (TFCs) could offer an alternative therapy; however, the minimal number of TFCs to be corrected remains unknown. Methods: Thyroid dyshormonogenesis in Duoxa-/- deficient mice was rescued by conditional thyroid-specific expression of DUOXA2/DUOX2 subunits. In order to restrict reactivation in a subset of TFCs, low doses of tamoxifen (0.1-2 mg) were injected. Thyroid function was assessed by immunostaining of iodinated-thyroglobulin (iTG). Circulating serum thyrotropin (TSH) and total thyroxin (T4) were quantified, and thyroidal expression of TSH-responsive genes (Nis, Tpo, Tshr) and hepatic deiodinase type-1 (Dio1) was measured. Last, combining iTG immunostaining with Duox2 in situ hybridization, we estimated the fraction of rescued TFCs required to restore follicular TG iodination. Results: Colloidal iTG+ staining in more than 90% of follicles demonstrated the successful rescue of TH biogenesis in doxycycline-induced Tet:Da2D2+/-;mTg:CreERT2+/-;Duoxa-/- animals (3TA). In contrast, reducing tamoxifen doses to 0.1 mg resulted in unresolved primary CH with thyroid enlargement, induction of Nis, Tpo, and Tshr, decrease of Dio1, and growth delay. Corresponding thyroid sections revealed scattered iTG+ colloidal lumens dispersed in histologically altered parenchyma. Nevertheless, we determined that only 11-15% of TFCs need to be reactivated within the follicle to functionally restore iodide organification. Goiter involution was also studied in 3TA following functional oxidase recovery or levothyroxine supplementation. Although thyroid enlargement was similarly reduced in both groups, expression of Nis, Tpo, Tshr, and Dio1 more rapidly normalized in genetically rescued 3TA. In these pathological conditions, around 50-70% of iTG+ follicles would seem sufficient to recover a healthy thyroid function until two weeks. Conclusions: Our data in Duoxa-/- mice suggest that the percentage of TFCs to be corrected is limited to 10-15% per follicle, which could be compatible with future somatic gene therapies. Furthermore, the reconstitution of hormonogenic-competent TFCs successfully results in goiter resolution, to an extent comparable to that of gold-standard TH replacement therapy.

Objective: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 10%–15% of pediatric and 25% of adult ALL cases. While intensified therapy has improved survival, outcomes for relapsed or refractory T-ALL remain poor, emphasizing the need to advance our understanding of T-ALL biology and develop precision oncology therapeutics. SOX11 is a transcription factor involved in embryogenesis and is aberrantly expressed in several tumor types. Although normally absent in thymocytes, SOX11 is expressed in a subset of T-ALL cases, where it may act as an oncogenic driver. Here, we explore the role of SOX11 in T-cell development and T-ALL initiation. Methods: To replicated elevated SOX11 levels, we generated a conditional Rosa26-SOX11 overexpression mouse model and crossed it with CD2-Cre or Lck-Cre drivers to express SOX11 in common lymphoid progenitors (SOX11CD2) or T-cells (SOX11Lck). These were further crossed with Lmo2 or MYCN models to obtain SOX11Lck;Lmo2CD2 and SOX11Lck;MYCNLck mice. CITE-seq was performed on spleens from 12-week-old control (n=3) and SOX11CD2 (n=3) mice. Thymi from 8-week-old SOX11Lck mice were analyzed to assess pre-leukemic changes. RNA-seq was conducted sorted CD4-CD8- double negative (DN) or CD4+CD8+ double negative (DP) cells from control, SOX11Lck, Lmo2CD2, and SOX11Lck;Lmo2CD2 mice. Thymic transplantation in sublethally irradiated mice was used to evaluate self-renewal of SOX11-overexpressing thymocytes. We analyzed publicly available transcriptomic data from 1,309 T-ALL patients to evaluate SOX11 expression and identify common genomic features in SOX11high T-ALL. Additionally, we transduced Lck-Cre+ bone marrow progenitors from control or SOX11Lck mice with either an empty vector or Cre-dependent MYCN-expression contstructs, and transplanted them into immunocompromised recipients to evaluate leukemogenic potential. Results: We analyzed the expression levels of SOX11 in 1309 T-ALL cases that were categorized according to the classifying driver or subtype. SOX11 was highly expressed in LMO1/2, TAL1/2, and KMT2A driver groups and in STAG&LMO2, KMT2A, LMO2 γδ-like, and TAL1 αβ-like subtypes. In contrast, SOX11 was low or absent in most thymocyte populations. scRNA-seq of SOX11CD2 mice revealed a block in αβ T-cell development and skewing toward B-cell and γδ T-cell fates, suggesting a potential role for SOX11 in LMO2 γδ-like T-ALL development, but also that SOX11 may exert its oncogenic role post T-cell commitment. To further dissect the role of SOX11 during T-cell development, we used a T-cell-restricted Lck-Cre driver in subsequent studies. Similar to a previously published Lmo2 model, SOX11Lck mice displayed an increase in immature DN3 thymocytes and a decrease of DP thymocytes. However, thymic transplantation assays demonstrated that, unlike Lmo2, SOX11 does not confer self-renewal capacity to thymocytes. Notably, co-expression of SOX11 and Lmo2 synergized to expand DN3 thymocytes and accelerate T-ALL onset, with median survival dropping from 332 days in Lmo2CD2 to 163 days in SOX11Lck;Lmo2CD2mice. To dissect downstream targets, we performed RNA-seq on FACS-sorted pre-leukemic and leukemic DN3 thymocytes. Enrichement analysis identified MYCN as a top SOX11 target. Strikingly, SOX11high T-ALL were enriched for MYCN P44L mutations (13/20 cases), which increase MYCN stability via disruption of a degradation motif. We validated MYCN as a functional SOX11 effector in genetic and transplantation models and found combined SOX11 and MYCN expression accelerated T-ALL onset. Conclusions: SOX11 is aberrantly expressed in a subset of T-ALLs and associated with MYCN P44L mutations. In mice, SOX11 induces a developmental arrest at the DN3 stage and synergizes with Lmo2 or MYCN to drive T-ALL formation.

Relapsed and refractory disease in children with T-cell acute lymphoblastic leukemia (R/R T-ALL) remains a major clinical challenge. Outcomes for children who relapse or exhibit resistance to initial treatments are dismal, with survival rates frequently below 25% despite aggressive therapy. To minimize toxicities and improve outcomes, individualized precision medicine approaches targeting the underlying biology of R/R T-ALL are especially important, considering that T-ALL is characterized by genetic, epigenetic and posttranscriptional heterogeneity, and organ and niche specificities (e.g. the central nervous system), all of which underlie disease progression and therapy resistance. Here, we summarize the current understanding of the complexity of pediatric T-ALL biology and how such knowledge may be clinically leveraged, emphasizing the need for innovative therapeutic routes to improve outcomes for children with R/R T-ALL. Emerging approaches that hold promise or show palpable results include proteasome inhibitors, BCL-2 antagonists, and JAK (for JAK- and IL-7R-driven cases), ABL and SRC family tyrosine kinase (for LCK-activated cases), MEK or PI3K-mTOR inhibitors. MYC-targeting agents, DNA demethylating agents, histone deacetylase inhibitors, splicing modulators, or drugs exploring T-ALL metabolic vulnerabilities, are other examples for potential pharmacological intervention. Immunotherapies, particularly CAR T-cell products targeting CD7 and other markers, but also biologics (e.g. targeting CD38), are under development and increasing interest. These agents should be rationally integrated into precision medicine combination therapies informed by genetic, epigenetic, and posttranscriptional insights that will be essential to refine risk stratification and minimize the risk of resistance. Novel strategies leveraging artificial intelligence and machine learning could accelerate discovery and optimize treatment frameworks.

Epithelial to Mesenchymal transitions (EMT) drive cell plasticity and are associated with cell features such as invasiveness, migration and stemness. They are orchestrated by select families of EMT-associated transcription factors, which exhibit pleiotropic roles in the malignant progression of various cancer types, such as breast and colorectal cancer (CRC). This has spurred interest in EMT as a promising target for the development of novel therapeutic strategies. In this study, we developed a phenotypic dual EMT Sensor screening assay, amendable to efficient high-throughput identification of small molecules interfering with EMT. In a proof-of-concept screening we identified anti-EMT repurposing drugs. From these, we validated RepSox, a selective inhibitor of the TGF-β type I receptor ALK5, and demonstrated that it is potently blocking EMT in both breast and colorectal cancer cell lines in vitro. In addition, utilizing a Drosophila melanogaster metastatic CRC model we confirmed the ability of the identified anti-EMT hits to suppress metastatic behavior in vivo.

In this study, hexamer peptide-based hydrogels were loaded with different model protein cargos and the release profiles investigated to explore the balance between injectability and loading capacity permitting the release of a therapeutically relevant dose. We demonstrate that the release of protein cargos from our hexamer peptide hydrogels depends on the stability of the hydrogel network, the mobility of the cargo to diffuse out of the network, and the interaction between the hydrogel network and the cargo. For the first time, our peptide hydrogels were used to develop an injectable sustained release formulation of a therapeutic enzyme, namely Erwinase®, an FDA-approved asparaginase for the treatment of acute lymphoblastic leukemia. We show that the current hexamer peptide-based hydrogels allow sufficient protein loading and sustained release of the fully active asparaginase enzyme both in vitro and in vivo. Altogether, this study describes how peptide hydrogels can be exploited to provide injectable slow-release formulations of biologics, including enzyme therapeutics, to enhance their clinical applicability.

RNA homeostasis is dysregulated in cancer and affects disease progression and therapy resistance. N6-methyladenosine (m6A), the most abundant epitranscriptomic modifica-tion in eukaryotic messenger RNA, plays a pivotal role in RNA biology, affecting transcript stability, translation, and splicing. Our study uncovers the extensive m6A changes in patients with T-cell acute lymphoblastic leukemia (T-ALL), to our knowledge, for the first time. It reveals m6A's regulatory role in the oncogenic MYC and cholesterol biosynthesis pathways. In addition, we discovered that T-ALL is highly dependent on the m6A reader heterogeneous nuclear ribonucleoprotein C (HNRNPC). HNRNPC is transcriptionally controlled by MYC and is an essential regulator of m6A-modified transcripts. Consequently, transcriptional silencing of HNRNPC profoundly impairs oncogenic pathways and critically diminishes leukemia cell growth. In addition, the levels of the m6A demethylase fat mass and obesity-associated protein (FTO) are significantly elevated in T-ALL cells compared with normal cells, and to other types of leukemia. Targeting FTO shows therapeutic potential in preclinical disease models and synergizes with clinically relevant therapeutics. Our findings underscore the integral role of RNA methylation in orchestrating cancer cell oncogene expression and metabolism and highlight promising novel therapeutic avenues for the treatment of T-cell leukemia.

L-asparaginase (ASNase) has been a cornerstone of pediatric acute lymphoblastic leukemia (ALL) treatment since the 1970s, leveraging a unique mechanism that exploits the metabolic vulnerability of leukemic cells. Cancer cells deficient in asparagine synthetase (ASNS), which rely on blood asparagine, are starved when ASNase depletes asparagine in the bloodstream. While this approach is effective in ALL, preclinical studies suggest that ASNase could also target other ASNS-deficient (ASNSlow) cancers, including acute myeloid leukemia (AML) and subsets of solid tumors like liver and colorectal cancers. However, the severe toxicity of existing ASNase formulations, largely due to off-target glutaminase activity and immunogenicity, has limited its broader clinical application. To overcome these barriers, we developed a novel human-like mammalian ASNase designed to minimize immunogenicity and eliminate glutaminase-driven toxicity while maintaining high specificity for asparagine. This engineered ASNase demonstrated robust in vitro and in vivo efficacy against both T-cell and B-cell ALL. Notably, it also showed potent activity as a monotherapy in AML, eradicating tumor cells in blood and bone marrow. Beyond blood cancers, this ASNase displayed significant anti-tumor effects against a CRISPR/CAS-9-engineered melanoma model with genetic loss of ASNS and a hepatocellular carcinoma model with low endogenous ASNS expression, achieving durable tumor suppression. This demonstrates that for ASNSlow tumors the glutaminase activity is not required for efficacy. Crucially, the novel ASNase was well tolerated in animal studies, enabling prolonged asparagine depletion—an essential factor for its striking anti-cancer effects. With its enhanced safety profile and specificity, this humanized ASNase represents a transformative advancement, extending the benefits of metabolic tumor starvation to a broader range of cancers. Its predictable mechanism of action, guided by biomarkers of ASNSlow status, positions it as a promising therapeutic breakthrough for targeting diverse malignancies. The authors used GPT-4.o in order to rephrase certain sentences. After using this tool or service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication. Amanda M. Schalk, Maaike Van Trimpont, Ying Su, Ashley De Loera, Alyssa Garcia, Tim Lammens, Steven Goossens, Arnon Lavie. A novel human-like asparaginase - indication expansion made possible [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 328.

Multiple Myeloma (MM) is a malignancy characterized by an uncontrolled proliferation of malignant plasma cells in the bone marrow and remains incurable. Treatment typically consists of a multimodal approach, with glucocorticoids (GC) as a crucial treatment pillar in the diagnosis and relapsed settings. Inevitably, patients become therapy resistant, but to which component of the treatment armamentarium the tumor becomes refractory is unknown. Here we used different in cellulo models of GC resistance to gain insights into the mechanistic processes of emerging GC therapy resistance. We found that differential baseline GC responsiveness of the cells is associated with significant differences in the timing and the degree to which myeloma cell lines become resistant to GCs. We corroborated that the chemokine receptor CCR1 is a shared biomarker between MM cell lines upon the emergence of GC resistance. Significant overlap exists between pathways enriched in partial GC-resistant MM.1S cells and those enriched in relapsed patients whose treatment included GCs. In addition, enrichment analyses demonstrated that alterations in metabolism and plasma cell expression signatures are associated with decreased sensitivity to GCs. From these analyses, we validated a biomarker, fatty acid synthase (FASN), and pinpointed its pivotal role in determining the GC sensitivity of myeloma cells, offering future opportunities for enforcement of GC sensitivity and re-sensitization.

TET2-mediated DNA demethylation plays a pivotal role in regulating pre-leukemic clonal expansion in acute myeloid leukemia (AML), where TET2 mutations are also linked to AML progression. However, its function in other types of leukemias, including T-cell acute lymphoblastic leukemia (T-ALL), remains unclear. Here, we used two different T-ALL mouse models to study the possible tumor suppressor role of Tet2 in pre-leukemic T-ALL. Overexpression of Tet2 resulted in a mild but significant increase in T-ALL latency in the immature CD2-Lmo2tg T-ALL mouse model, but no effect on survival was observed in the mature Lck-Cretg/+ Ptenfl/lf T-ALL mouse model. In contrast to the pre-leukemic thymocytes from CD2-Lmo2tg mice, Lck-Cretg/+ Ptenfl/fl thymi do not display self-renewal suggesting that the anti-leukemic effect of Tet2 occurs mainly in the pre-leukemic phase of T-ALL. In conclusion, we demonstrated that the Tet2 tumor suppressor function is dependent on the differentiation stage of T-ALL and limited to the pre-leukemic phase.

Current therapies for neuroblastoma are often ineffective and survivors suffer from severe long-term therapy related side-effects, underscoring the need for identification of novel drugging strategies. We performed an in-depth evaluation of phenotypic and molecular responses following exposure of neuroblastoma cells to the rocaglate CR-1-31-B, scrutinizing its mode-of-action through integrative ribosome footprinting and shotgun proteome profiling. We could show that CR-1-31-B significantly reduces tumor growth in vivo without apparent toxicity. By means of combined ribosome footprinting and transcriptome analysis we uncovered that CR-1-31-B treatment downregulates translation efficiencies of several major neuroblastoma dependencies including MYCN, CCND1 and ALK as well as factors involved in the G2/M checkpoint. Upregulated targets are enriched for oxidative phosphorylation pathway components and DNA repair. At the proteome level, CR-1-31-B imposed downregulation of a FOXM1 driven signature, including the FOXM1 target gene TPX2. We show that neuroblastoma cells are dependent on TPX2 for growth and DNA repair and further demonstrate enhanced CHK1 sensitivity upon TPX2 knockdown. Next, we also observed synergistic effects of CHK1 inhibition with CR-1-31-B. In conclusion, our data support CR-1-31-B as a potent novel therapeutic agent in neuroblastoma, in particular in combination with DNA damage or replication stress inducing agents. ### Competing Interest Statement The authors have declared no competing interest.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy. Current intensified therapeutic protocols coincide with severe side effects, and no salvage therapy is available for primary therapy-resistant or relapsed patients. This highlights the need to identify new therapeutic targets in T-ALL. PSIP1, dispensable for normal hematopoiesis, is a dependency factor in KMT2A -rearranged myeloid leukemia. Nonetheless, loss-of-function mutations suggest a tumor suppressor role for PSIP1 in T-ALL. Here, we demonstrate that the loss of Psip1 accelerates T-ALL initiation in mice which we correlated with reduced H3K27me3 binding. Contrastingly, loss of PSIP1 impaired cell proliferation in several T-ALL cell lines. In cell lines, PSIP1 down-regulation leads to a reduction of COX20, an assembly factor of the cytochrome c oxidase in the mitochondria, and to a reduction in mitochondrial respiration. This indicates that PSIP1 can exert a dual role in the context of T-ALL, either as a tumor suppressor gene during tumor initiation or as a dependency factor in tumor maintenance.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by the clonal expansion of immature T-cell progenitors. The pathogenesis is complex and involves the dysregulation of multiple signaling pathways and transcription factors essential for T-cell development. Early T-cell precursor ALL (ETP-ALL) is a distinct molecular subclass of T-ALL characterized by an early T-cell differentiation arrest with myeloid and stem cell properties.We report the identification of a CDK6-IKZF1 fusion in an ETP-ALL patient. Based on detailed characterization of this fusion product, we hypothesized that loss of IKZF1 is the true oncogenic driver in this ETP-ALL patient. As 5-15% of ETP-ALL patients harbor IKZF1 deletions, we evaluated the impact of Ikzf1 loss alone or in combination with Lmo2 overexpression, which results in the spontaneous development of immature T-ALL in mice. Our results demonstrate that heterozygous loss of Ikzf1 collaborates with gain of Lmo2, whilst homozygous loss of Ikzf1 on its own is sufficient to induce murine T-ALL, independently of Lmo2 overexpression. Next, we evaluated whether loss of Ikzf1 induces self-renewal in pre-leukemic thymocytes, similar as has previously been demonstrated for Lmo2 overexpressing thymocytes. Our results clearly show that Ikzf1 deleted thymocytes can repopulate and self-renew the thymus, while wildtype thymocytes do not.In conclusion, our results indicate that Ikzf1 is a tumour suppressor in ETP-ALL and its loss results in the acquisition of aberrant preleukemic thymic self-renewal properties. Since pre-leukemic thymocytes have been demonstrated to be more chemotolerant and a potential source of relapse, further molecular insights on how self-renewal is regulated upon Ikzf1 loss, could provide valuable input for the development of novel therapeutic strategies in ETP-ALL.

Thyroid cancer is the most common endocrine malignancy and several genetic events have been described to promote the development of thyroid carcinogenesis. Besides the effects of specific mutations on thyroid cancer development, the molecular mechanisms controlling tumorigenesis, tumor behavior, and drug resistance are still largely unknown. Cancer organoids have been proposed as a powerful tool to study aspects related to tumor development and progression and appear promising to test individual responses to therapies. Here, using mESC-derived thyroid organoids, we developed a BrafV637E-inducible model able to recapitulate the features of papillary thyroid cancer in vitro. Overexpression of the murine BrafV637E mutation, equivalent to BrafV600E in humans, rapidly triggers to MAPK activation, cell dedifferentiation, and disruption of follicular organization. BrafV637E-expressing organoids show a transcriptomic signature for p53, focal adhesion, ECM-receptor interactions, EMT, and inflammatory signaling pathways. Finally, PTC-like thyroid organoids were used for drug screening assays. The combination of MAPK and PI3K inhibitors reversed BrafV637E oncogene-promoted cell dedifferentiation while restoring thyroid follicle organization and function in vitro. Our results demonstrate that pluripotent stem cells-derived thyroid cancer organoids can mimic tumor development and features while providing an efficient tool for testing novel targeted therapies.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.