Acute myeloid leukemia (AML) with complex karyotype is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. Genomic rearrangements can reposition active enhancers near proto-oncogenes, leading to their aberrant expression; however, a comprehensive understanding of these events in AML is still incomplete. To facilitate the discovery of such "enhancer hijacking" events, we developed Pyjacker, a computational tool, and applied it to 39 AML samples with complex karyotype. Pyjacker identified several enhancer hijacking events in AML patient samples, including aberrant expression of MNX1, which can result from del(7)(q22q36) and is associated with hijacking of a CDK6 enhancer. MNX1 activation occurred in 1.4% of patients with AML and showed significant co-occurrence with BCOR mutations. Through a xenograft mouse model, we demonstrated that MNX1 is required for leukemia cell fitness. Pyjacker is an easy-to-use, accurate, and broadly applicable tool for identifying consequences of genomic events driving tumorigenesis, especially when germline genomic data are missing. SIGNIFICANCE:This study examines the consequences of structural alterations in AML and demonstrates that proto-oncogene activation by enhancer hijacking is an understudied pathomechanism. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency but also to activation of oncogenes by enhancer hijacking.

CASZ1 is a conserved transcription factor involved in neural development, blood vessel assembly and heart morphogenesis. CASZ1 has been implicated in cancer, either suppressing or promoting tumor development depending on the tissue. However, the impact of CASZ1 on hematological tumors remains unknown. Here, we show that the T-cell oncogenic transcription factor TAL1 is a direct positive regulator of CASZ1, that T-cell acute lymphoblastic leukemia (T-ALL) samples at diagnosis overexpress CASZ1b isoform, and that CASZ1b expression in patient samples correlates with PI3K-AKT-mTOR signaling pathway activation. In agreement, overexpression of CASZ1b in both Ba/F3 and T-ALL cells leads to the activation of PI3K signaling pathway, which is required for CASZ1b-mediated transformation of Ba/F3 cells in vitro and malignant expansion in vivo. We further demonstrate that CASZ1b cooperates with activated NOTCH1 to promote T-ALL development in zebrafish, and that CASZ1b protects human T-ALL cells from serum deprivation and treatment with chemotherapeutic drugs. Taken together, our studies indicate that CASZ1b is a TAL1-regulated gene that promotes T-ALL development and resistance to chemotherapy.

Acute myeloid leukemia with complex karyotype (ckAML) is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. We hypothesized that the numerous genomic rearrangements could reposition active enhancers near proto-oncogenes, leading to their aberrant expression. We developed pyjacker, a computational tool for the detection of enhancer hijacking events, and applied it to a cohort of 39 ckAML samples. Pyjacker identified motor neuron and pancreas homeobox 1 ( MNX1 ), a gene aberrantly expressed in 1.4% of AML patients, often as a result of del([7][1])(q22q36) associated with hijacking of a CDK6 enhancer. MNX1 -activated cases show significant co-occurrence with BCOR mutations and a gene signature shared with t(7;12)(q36;p13) pediatric AML. We demonstrated that MNX1 is a dependency gene, as its knockdown in a xenograft model reduces leukemia cell fitness. In conclusion, enhancer hijacking is a frequent mechanism for oncogene activation in AML. Statement of significance This study examines the consequences of structural alterations and demonstrates that proto-oncogene activation by enhancer hijacking is an overlooked pathomechanism in AML. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency, but also to activation of oncogenes by enhancer hijacking, providing a novel leukemogenic mechanism. ### Competing Interest Statement UHT is currently employed at Oxford Nanopore Technologies. EJ is currently employed at AstraZeneca. LB has received honoraria from AbbVie, Amgen, Astellas, BristolMyers Squibb, Celgene, Daiichi Sankyo, Gilead, Hexal, Janssen, Jazz Pharmaceuticals, Menarini, Novartis, Pfizer, Roche, and Sanofi, as well as research support from Bayer and Jazz Pharmaceuticals. DBL received honoraria from Infectopharm GmbH. All other authors declared no conflict of interest. [1]: #ref-7

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Acute Myeloid Leukemia (AML) is a highly aggressive adult leukemia with a high incidence of relapse and mortality. AML hijacked the hierarchical organization of normal hematopoiesis with Leukemia Stem Cells (LSCs) at the top of the differentiation tree and differentiated and highly proliferating myeloid blast at the bottom. Similar to normal hematopoietic stem cells (HSCs), LSCs have been originally identified as CD34+CD38- cells. However, this strategy to isolate LSCs is particularly challenging in NPM1 mutated AMLs, since expression of CD34 on leukemic cells is low or absent altogether. Aims: In this study, we aimed to establish a novel sorting strategy to isolate LSCs independently of CD34 surface marker in both CD34positive and CD34negative AMLs. Methods: Using a genetically highly homogeneous cohort of 46 AML patients carrying DNMT3A and NPM1 mutations, we established a novel isolation strategy to enrich for LSCs even in CD34negative AMLs by additionally including a positive selection for GPR56 and a negative selection for mature markers such as NKG2DLigandss. Results: By combining functional in vivo data, mutational analyses on xenografts and transcriptomic data, we found that CD34+GPR56+NKG2DL- subpopulations can contain along with LSCs also normal and/or pre-leukemic (HSCs). In contrast, no such residual normal or pre-leukemic HSCs were detected in CD34-GPR56+NKG2DL- subpopulations. Finally, we show that naïve AML patients who retain a relevant reserve of normal and/or preleukemic HSCs, able to reconstitute immunocompromised mice at time of diagnosis, have significant longer relapse-free and overall survival compared to patients without engrafting HSCs. Summary/Conclusion: These data suggest that the presence of normal and/or preleukemic HSCs with multilineage engrafting potential in the bone marrow of naïve patients is associated with good response to standard chemotherapy, providing a basis for the development of novel predictive biomarkers for standard chemotherapy regime. Keywords: DNMT3A, AML, Leukemic stem cell, Stem cell marker

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of relapse and mortality. Current chemotherapies whilst successful in eradicating blasts, are less effective in eliminating relapse-causing leukemic stem cells (LSCs). Although LSCs are usually identified as CD34+CD38- cells, there is significant heterogeneity in surface marker expression, and CD34- LSCs exist particularly in NPM1mut AMLs. By analyzing diagnostic primary DNMT3AmutNPM1mut AML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34neg AML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D ligands are used. We show that the functional reconstitution capacity of CD34- and CD34+ LSCs as well as their transcriptomes are very similar which support phenotypic plasticity. Furthermore, we show that although CD34+ subpopulations can contain next to LSCs also normal and/or preleukemic hematopoietic stem cells (HSCs), this is not the case in CD34-GPR56+NKG2DL- enriched LSCs which thus can be isolated with high purity. Finally, we show that patients with AML, who retain at the time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significantly longer relapse-free and overall survival than patients with AML in whom functional HSCs are no longer detectable.

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4+ T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4+ T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool.

Acute myeloid leukemia (AML) is a very heterogeneous and highly malignant blood cancer. Mutations of the DNA methyltransferase DNMT3A are among the most frequent recurrent genetic lesions in AML. The majority of DNMT3A -mutant AML patients shows fast relapse and poor survival, but also patients with long survival or long-term remission have been reported. Underlying molecular signatures and mechanisms that contribute to these survival differences are only poorly understood and have not been studied in detail so far. We applied hierarchical clustering to somatic gene mutation profiles of 51 DNMT3A -mutant patients from The Cancer Genome Atlas (TCGA) AML cohort revealing two robust patient subgroups with profound differences in survival. We further determined molecular signatures that distinguish both subgroups. Our results suggest that FLT3 and/or NPM1 mutations contribute to survival differences of DNMT3A -mutant patients. We observed an upregulation of genes of the p53, VEGF and DNA replication pathway and a downregulation of genes of the PI3K-Akt pathway in short- compared to long-lived patients. We identified that the majority of measured miRNAs was downregulated in the short-lived group and we found differentially expressed microRNAs between both subgroups that have not been reported for AML so far ( miR-153-2 , miR-3065 , miR-95 , miR-6718 ) suggesting that miRNAs could be important for prognosis. In addition, we learned gene regulatory networks to predict potential major regulators and found several genes and miRNAs with known roles in AML pathogenesis, but also interesting novel candidates involved in the regulation of hematopoiesis, cell cycle, cell differentiation, and immunity that may contribute to the observed survival differences of both subgroups and could therefore be important for prognosis. Moreover, the characteristic gene mutation and expression signatures that distinguished short- from long-lived patients were also predictive for independent DNMT3A -mutant AML patients from other cohorts and could also contribute to further improve the European LeukemiaNet (ELN) prognostic scoring system. Our study represents the first in-depth computational approach to identify molecular factors associated with survival differences of DNMT3A -mutant AML patients and could trigger additional studies to develop robust molecular markers for a better stratification of AML patients with DNMT3A mutations.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy in which the transformed clone is arrested during T-cell development. Several genetic and epigenetic events have been implicated in this transformation. MicroRNAs (miRNAs) are small, non-coding RNAs that primarily function as endogenous translational repressors of protein-coding genes. The involvement of miRNAs in the regulation of cancer progression is well-established, namely by down-regulating the expression of key oncogenes or tumor suppressors and thereby preventing or promoting tumorigenesis, respectively. Similar to other cancers, several miRNA genes have been identified and implicated in the context of T-ALL. In this review we focused on the most studied microRNAs associated with T-ALL pathogenesis.

Leukemic stem cells were hypothesized to play a critical role in acute myeloid leukemia relapse, but data to support this were lacking. In a recent study elegantly combining sequencing with functional xenograft assays, Shlush et al ( ) identified two distinct origins of leukemic relapse. They provided direct experimental evidence linking relapse to cancer stem cell clones already present before therapeutic intervention.

Previous results indicated that miR-146b-5p is downregulated by TAL1, a transcription factor critical for early hematopoiesis that is frequently overexpressed in T-cell acute lymphoblastic leukemia (T-ALL) where it has an oncogenic role. Here, we confirmed that miR-146b-5p expression is lower in TAL1-positive patient samples than in other T-ALL cases. Furthermore, leukemia T-cells display decreased levels of miR-146b-5p as compared to normal T-cells, thymocytes and other hematopoietic progenitors. MiR-146b-5p silencing enhances the in vitro migration and invasion of T-ALL cells, associated with increased levels of filamentous actin and chemokinesis. In vivo, miR-146b overexpression in a TAL1-positive cell line extends mouse survival in a xenotransplant model of human T-ALL. In contrast, knockdown of miR-146b-5p results in leukemia acceleration and decreased mouse overall survival, paralleled by faster tumor infiltration of the central nervous system. Our results suggest that miR-146b-5p is a functionally relevant microRNA gene in the context of T-ALL, whose negative regulation by TAL1 and possibly other oncogenes contributes to disease progression by modulating leukemia cell motility and disease aggressiveness.

The transcription factor TAL1 is a proto-oncogene whose aberrant expression in committed T-cell precursors is associated with the development of T-cell acute lymphoblastic leukemia (T-ALL). The mechanisms leading to aberrant activation of TAL1 in T-ALL patients who lack chromosomal rearrangements involving the TAL1 locus remain largely unknown. We hypothesized that TAL1 levels decrease during normal T-cell development at least in part due to miRNA-dependent silencing, in which case TAL1 over-expression in some T-ALL cases could be the consequence of deregulated miRNA expression. By performing computational prediction of miRNAs that bind to the human TAL1 mRNA we compiled a list of miRNAs that are candidates to regulate TAL1. Using a luciferase reporter system and mutagenesis assays we confirmed the miRNA-TAL1 mRNA interactions and selected candidate miRNAs: miR-101, miR-520d-5p, miR-140-5p, miR-448 and miR-485-5p. Over-expression of these microRNAs in different T-ALL cell lines consistently resulted in the down-regulation of TAL1 protein. In accordance, inhibition of miR-101 and miR-520d-5p promoted TAL1 protein expression. Importantly, we found that miR-101, miR-140-5p, miR-448 and miR-485-5p were down-regulated in T-ALL patient specimens and T-ALL cell lines. Our results show for the first time the existence of epigenetic regulation of TAL1 by specific miRNAs which may contribute, at least in part, to the ectopic expression of TAL1 in some T-ALL cases.

TAL1/SCL/TCL5 is a critical transcription factor for hematopoietic stem cell maintenance and regulation of early hematopoiesis. However, aberrant expression of TAL1 in committed T-cell precursors is also directly implicated in the development of T-cell leukemia. Roughly 25 years ago TAL1 was identified in early hematopoietic cells and involved in leukemia. Here, we review the wealth of knowledge gained since then on its physiological roles and mechanisms by which TAL1 ectopic expression contributes to leukemogenesis. We emphasize recent findings that shed light into the intricacies of TAL1 (epi) genetic regulation and the transcription network orchestrated by this major T-cell oncogene. Importantly, an exciting time is coming when data using the mechanistic knowledge accumulated on TAL1 may be used to develop novel anti-leukemia targeted therapies.

Mutations and deletions of the tumour suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) are frequently involved in the development of cancer. However, PTEN is also tightly controlled by various non-genomic mechanisms. This review focuses on those mechanisms, namely on the epigenetic silencing of PTEN, post-transcriptional regulation by non-coding RNAs and post-translational modification. We summarise their involvement in cancer in general, and place some emphasis on leukaemia, where PTEN genetic lesions are relatively uncommon and, strikingly, high levels of PTEN expression frequently associate with PTEN functional inactivation. Overall, it is apparent that rather than looking strictly for PTEN genetic lesions and PTEN expression status, the key to evaluating the real impact of PTEN as a ‘quasi-insufficient’ tumour suppressor must rely on the complete understanding of PTEN’s ‘functional dose’, incorporating the multiple layers of PTEN regulation in the cell that are ultimately compromised in a given cancer.