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Currently, the limited capacity of pediatric acute myeloid leukemia (AML) therapies to prevent recurrence has contributed to high mortality rates. While dormant self-renewing leukemia stem cells (LSCs) contribute to adult AML relapse, their role in pediatric AML therapeutic resistance has not been clearly elucidated and thus was investigated in the context of this study. Through whole transcriptome sequencing (RNA-seq) analyses of FACS-purified human hematopoietic stem cells (HSCs; CD34 +CD38 -Lineage -) and progenitor cells (HPCs; CD34 +CD38 + Lineage -) from pediatric AML (n=10) compared with adult de novo (n=5) and secondary AML (n=6) as well as non-leukemic pediatric bone marrow samples (n=6), we identified widespread splicing alterations in pediatric AML compared to non-leukemic donors, indicative of a disruption in splicing regulation. In this study, we identified 2,000 exon skipping events in pediatric AML HSCs and HPCs. Moreover, we detected increased exon skipping and intron retention in stem cell self-renewal and survival transcripts in pediatric AML stem and progenitor cells. Specifically, the pro-survival isoform of MCL1, MCL1 long, was significantly increased in comparison to its pro-apoptotic counterpart, MCL1 short. In addition, self-renewal, RNA editing and splice isoform altering adenosine deaminase RNA specific 1 (ADAR1) p150 isoform levels were significantly (p=0.05) upregulated in pediatric AML progenitors suggesting that splicing and RNA editing deregulation could fuel pediatric AML stem and progenitor cell propagation. After successful completion of pre-IND development of a pharmacologically stable, potent, and selective small molecule splicing modulator, Rebecsinib (17S-FD-895) (Crews, Balain et al Cell Stem Cell 2016; Chan et al Cell Reports 2020), we developed a dual fluorescence lentiviral splicing reporter that assays the on target anti-leukemic efficacy of Rebcsinib and to assess the therapeutic index between LSCs and normal hematopoietic stem and progenitor cells. In hematopoietic progenitor assays, we observed a dose-dependent reduction in clonogenicity and replating of CD34 + cells isolated from pediatric AML samples following treatment with Rebecsinib. While pediatric AML samples were more sensitive to splicing modulation than adult de novo or adult secondary AML samples, normal cord blood progenitor samples were unaffected by splicing modulator treatment. In addition, we identified dose-dependent alterations in lentiviral splicing reporter activity in pediatric leukemia cells engrafted in a humanized AML mouse xenograft model following intravenous treatment with one dose of 10mg/kg and 20mg/kg of Rebecsinib. Finally, we observed a reduction in ADAR1 p150 transcripts by RNA-seq analysis of hematopoietic tissues in serially transplanted patient derived AML xenografts after Rebecsinib treatment suggesting that inhibition of ADAR1 splicing prevents LSC self-renewal. Cumulatively, these data demonstrate that stem and progenitor cell specific deregulation of pre-mRNA splicing and ADAR1 activation represent a therapeutic vulnerability to splicing modulation, which provides a strong rationale for developing Rebecsinib for preventing pediatric AML recurrence. Disclosures Cloos: Astellas: Speakers Bureau; DC-One: Other, Research Funding; Genentech: Research Funding; Helsinn: Other; Janssen: Research Funding; Merus: Other, Research Funding; Navigate: Patents & Royalties; Novartis: Consultancy, Other, Research Funding; Takeda: Research Funding. Crews: Ionis Pharmaceuticals: Research Funding. Burkart: Algenesis: Other: Co-founder. Jamieson: Forty Seven Inc.: Patents & Royalties.

Background Childhood cancer survivors are confronted with a variety of chronic health conditions as a consequence of their life saving therapy. Chemotherapy is the cornerstone of childhood cancer treatment, which consists of combinations of various drugs administered over multiple years. Most chemotherapeutic drugs act by fatally damaging the DNA or blocking the replication thereof. Although high-intensity chemotherapy efficiently eradicates tumor cells, the impact on the genomes of normal, that is noncancerous, cells remains unknown. Mutagenicity of cancer treatment on normal hematopoietic cells may contribute to the development of chronic health conditions later in life, such as therapy-related acute myeloid leukemia (t-AML). Here, we characterized the mutational consequences of chemotherapy in the genomes of hematopoietic stem and progenitor cells (HSPCs) of children treated for cancer. Methods This study was approved by the Biobank and Data Access Committee of the Princess Máxima Center for Pediatric Oncology and all samples were obtained via the in-house biobank. We performed whole-genome sequencing (WGS) on multiple single HSPCs from all patients as well as the t-AML if available. In case of t-AML patients, samples were taken before t-AML treatment. Data on mutation accumulation in HSPCs of healthy individuals from 0-63 years of age was previously acquired. To distinguish the effects of different chemotherapies, we developed an in vitro approach using cord blood HSPCs to experimentally define the mutational consequences of individual chemotherapeutic drugs in primary human cells. Results Our cohort consisted of 22 pediatric cancer patients of which we obtained bone marrow aspirates and/or peripheral blood from timepoints pre- and post-treatment for their first cancer. We included 17 t-AML patients with varying first cancer diagnoses and five acute lymphoblastic leukemia (ALL) patients who did not develop t-AML. Patient characteristics and therapy information is described in Table 1. We compared the mutational burden of pre- and post-treatment HSPCs of the childhood cancer patients to those of healthy treatment-naïve individuals. HSPCs at time of first diagnosis of childhood cancer patients displayed a mutation burden similar to HSPCs of healthy individuals. In contrast, post-treatment HSPCs showed a significantly higher increase in mutation number over time than expected by normal ageing. We used mutational signature analysis to pinpoint the causes of this increased mutation burden in post-treatment HSPCs. Surprisingly, in most of these HSPCs the additive mutational effect could be attributed to clock-like processes, active during normal aging. Only few chemotherapeutic drugs showed direct mutagenic effects, such as platinum drugs and thiopurines. Whereas cisplatin-induced mutations could be observed in all cells exposed to this drug, thiopurine-induced mutations were present in a subset of the exposed HSPCs. These differences in thiopurine-induced mutagenesis across multiple exposed HSPCs could even be observed within individual patients. In one patient, the HSPCs containing the thiopurine-signature shared the oncogenic MLL-rearrangement with the t-AML blasts and had shorter telomeres compared to the HSPCs without this signature. This finding indicates that these cells have undergone more cell divisions, suggesting that thiopurine-induced mutagenesis requires DNA replication. We also identified novel therapy-associated mutational signatures. One of these signatures was observed in multiple patients who received a hematopoietic stem cell transplantation, as part of their cancer treatment, after which they displayed a viral reactivation for which they were treated. By WGS of in vitro exposed cord blood HSPCs, we demonstrated that this signature is caused by the antiviral drug ganciclovir, which is commonly used to treat cytomegalovirus infections. The second signature was present in HSPCs of a patient who received a combination of thiotepa and treosulfan. Conclusions Enhanced mutation accumulation after pediatric cancer treatment is caused by both direct and indirect mechanisms. The variance in mutagenic effects of (chemo)therapies on healthy HSPCs may influence the risk an individual patient has to develop t-AML and could, in future, play a role in t-AML risk assessment of pediatric cancer survivors and the development of new treatment regimens. Figure 1 Figure 1. Disclosures Zwaan: Sanofi: Consultancy.

The forkhead transcription factor FOXP1 is involved in B-cell development and function and is generally regarded as an oncogene in activated B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue lymphoma, lymphomas relying on constitutive nuclear factor κB (NF-κB) activity for survival. However, the mechanism underlying its putative oncogenic activity has not been established. By gene expression microarray, upon overexpression or silencing of FOXP1 in primary human B cells and DLBCL cell lines, combined with chromatin immunoprecipitation followed by next-generation sequencing, we established that FOXP1 directly represses a set of 7 proapoptotic genes. Low expression of these genes, encoding the BH3-only proteins BIK and Harakiri, the p53-regulatory proteins TP63, RASSF6, and TP53INP1, and AIM2 and EAF2, is associated with poor survival in DLBCL patients. In line with these findings, we demonstrated that FOXP1 promotes the expansion of primary mature human B cells by inhibiting caspase-dependent apoptosis, without affecting B-cell proliferation. Furthermore, FOXP1 is dependent upon, and cooperates with, NF-κB signaling to promote B-cell expansion and survival. Taken together, our data indicate that, through direct repression of proapoptotic genes, (aberrant) expression of FOXP1 complements (constitutive) NF-κB activity to promote B-cell survival and can thereby contribute to B-cell homeostasis and lymphomagenesis.

Children with Down syndrome are predisposed to leukemia during the first years of their life. 5-10% of newborns with Down syndrome are born with transient myeloproliferative disorder (TMD), which often spontaneously disappears. The majority of these patients achieves complete remission. However, in 20-30% of all TMD patients the disease progress in acute megakaryoblastic leukemia. In addition, they have a 20 fold higher risk of developing B-lymphocyte acute leukemia (B-ALL). Leukemic development in Down syndrome is initiated during fetal development. However, it is unclear why fetuses with a trisomy of chromosome 21 have an increased risk of developing leukemia. Previously, we have developed a method to study somatic mutations in single cells using clonal cultures. Here, we applied this method to human fetal hematopoietic stem and progenitor cells (HSPCs) from liver and bone marrow of Down syndrome human fetuses and control fetuses with two copies of chromosome 21 (D21). In addition, we characterized somatic mutation accumulation in not affected small intestine stem cells. Subsequently, we performed in depth mutational analyses to characterize active processes using mutational signatures in fetal stem cells, which potentially can drive leukemic development during early life. Recently, we have shown that that healthy adult HSPCs gradually accumulate somatic mutations in a linear fashion with an annual mutation rate of 14.2 base substitutions per year. Whereas the somatic mutation rate is significantly higher during fetal development. Subsequently, in Down syndrome fetuses the overall somatic mutation rate of fetal stem cells is significantly increased compared to D21 fetal stem cells (P-value: 0,024). We performed phylogenetic analysis to study relatedness of the cells and observed an higher somatic mutation rate in the first cell divisions. This elevated mutation rate can be explained by increased contribution of mutational process signature 1 and 5, which are already present in fetal stem cells. Therefore, Down syndrome fetal stem cells show enhanced activity or increased sensitivity to mutational processes that are normally active during development. The same mutational signatures are present in TMD blast cells, indicating that these processes can cause cancer driver mutations and subsequently contribute to leukemic development. Interestingly, some Down syndrome fetal stem cells showed very high mutation numbers that could partly be attributed to mutational signature 18, which likely reflect oxidative-stress induced mutagenesis. These findings, show increased mutagenesis in Down syndrome during fetal development in hematopoietic stem cells and small intestine stem cells. This increased mutagenesis can potentially explain why children with Down syndrome have an increased risk of developing leukemia in early life. Disclosures No relevant conflicts of interest to declare.

Background: T-cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset of ALL, the most frequent childhood malignancy. Although risk-adjusted chemotherapeutic regimens are currently extremely effective, their efficacy is associated with significant long-term side effects. Moreover, a significant fraction of the patients still relapse despite intensive chemotherapy, prompting the need for a deeper understanding of T-ALL biology in order to develop novel therapies. Interleukin-7 is a cytokine essential for normal T-cell development, where it has a pivotal role in promoting thymocyte survival via Bcl-2 upregulation. In this normal setting, Bcl-2 is under the control of STAT5 mediated signaling. Previously, we have shown that IL-7 promotes T-ALL expansion in vivo and leukemia cell survival and proliferation in vitro by activating PI3K/Akt/mTOR signaling pathway, consequently leading to p27kip1 downregulation and Bcl-2 upregulation. However, it is also known that T-cell lymphomas arising spontaneously in IL-7 transgenic mice depend on STAT5 activity and that leukemias displaying IL7R gain-of-function mutations are sensitive to JAK and STAT5 inhibitors. Thus, we investigated whether STAT5 could also be involved in the IL-7 pro-leukemia effects in human T-ALL cells. Methods: We used an IL-7-dependent leukemia T-cell line (TAIL7), an IL-7-responsive T-ALL cell line (HPB-ALL, with or without shRNA-mediated STAT5 silencing), primary T-ALL samples collected at diagnosis and patient-derived xenografts (PDX) and treated them with inhibitors of STAT5 (N-((4-Oxo-4H-chromen-3-yl) methylene) nicotinohydrazide) and PIM (AZD1208). Analysis of viability, cell size, cell cycle, surface CD71 and Bcl-2 expression was performed by flow cytometry. Signaling pathway activation, STAT5, PIM1, Bcl-xL, Mcl-1 and cell cycle protein expression was performed by immunoblot analysis. Proliferation was assessed by 3H-Thymidine incorporation. STAT5 ChIP-seq and RNA-seq were performed on TAIL7 cells. ChIP-PCR of histone marks H3K4me3, H3K27me, H3K27ac was performed in TAIL7 and HPB-ALL. Results: IL-7 induces JAK/STAT5 pathway activation in T-ALL cells and STAT5 genetic or pharmacological inactivation prevents IL-7-mediated T-ALL cell viability, growth and proliferation. At the molecular level, STAT5 is required for IL-7-induced downregulation of p27kip1, and upregulation of Cyclin A and TfR/CD71. Surprisingly, STAT5 inhibition does not significantly affect IL-7-mediated Bcl-2 upregulation, suggesting that, contrary to normal T-cells, STAT5 promotes leukemia cell survival via a Bcl-2-independent mechanism. In addition, IL-7-mediated increase in transcription of BCL2, BCL2L1 (Bcl-xL) and MCL1 is not affected by STAT5 silencing. To understand how STAT5 mediates the survival effects of IL-7 in T-ALL cells without affecting BCL2 transcription, we performed STAT5 ChIP-seq together with RNA-seq. Data cross-analysis reveal a diverse IL-7-driven STAT5-dependent transcriptional program in T-ALL cells, which includes transcription of the serine/threonine kinase PIM1. PIM1 is involved in cell cycle regulation and apoptosis, thereby constituting a possible alternative to Bcl-2-dependent prevention of apoptosis. We show that STAT5 silencing prevents IL-7-mediated PIM1 expression and the upregulation of active chromatin marks, H3K4me3 and H3K27ac, at the STAT5 binding region in the PIM1 gene. Notably, pharmacological inhibition of PIM kinase abrogates IL-7-mediated T-ALL cell growth and proliferation, however, without affecting cell survival. In agreement, PIM inhibition does not affect expression of Bcl-2 or Bcl-2 family anti-apoptotic members Bcl-xL and MCL1. Conclusion: Here we present evidence that T-ALL cells may have an alternative wiring of signaling networks downstream of IL-7 to that present in normal T-cells. In contrast to healthy lymphoid cells, IL-7-mediated control of survival of T-ALL cells via STAT5 does not rely on modulation of Bcl-2. Moreover, exploration of STAT5 downstream signaling reveals that PIM1 is required for IL-7-mediated proliferation of human T-ALL cells, indicating that strategies involving the use of PIM kinase small molecule inhibitors may have therapeutic potential against leukemias that rely on IL-7R and STAT5 signaling. Disclosures Barata: Instituto de Medicina Molecular João Lobo Antunes: Patents & Royalties: Patents.

Expression of the forkhead transcription factor FOXP1 is essential for early B-cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B-cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B-cell lymphoma and mucosa-associated lymphoid tissue lymphoma, being associated with poor prognosis. Here, by gene expression analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation and sequencing, we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs, but expression strongly decreases during PC differentiation. Moreover, as compared with immunoglobulin (Ig) M(+) MBCs, IgG(+) MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B-cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant expression of FOXP1 might contribute to lymphomagenesis by blocking this terminal B-cell differentiation.