Pediatric Acute Megakaryoblastic Leukemia Research Articles

A retrospective analysis of gene fusions and treatment outcomes in pediatric acute megakaryoblastic leukemia without Down syndrome.

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A Multicenter Clinical Study with Reduced Intensive Chemotherapy for Induction Remission of Pediatric Acute Megakaryoblastic Leukemia

Bcakground: Pediatric acute megakaryoblastic leukemia (AMKL) without Down syndrome (DS) is a genetically heterogenous myeloid malignancy and with dismal prognosis. Refractory and relapse remains a major challenge. Given to the young age distribution of AMKL and high frequency of abandonment in middle-income countries, we conducted a multicenter clinical trial (ChiCTR1800015875) in aims to lower the intensity of induction while without compromise the remission rate and survival. Methods: From June 2018 to December 2022, there were consecutively 43 cases with de novo non-DS AMKL, which accounted for 7.6% cases of acute myeloid leukemia (AML) during the same period, enrolled in this trial. All the patients received at least one cycle of low-dose induction with HAG regimen (homoharringtonine 1 mg/m 2, intravenous daily, days 1-7; cytarabine 10 mg/m 2, subcutaneously every 12 hours, 20 doses, and G-CSF 5µ/kg subcutaneous, daily, 10 doses) followed by 3 to 4 courses of intensive chemotherapy or HSCT as post-remission consolidation. Besides conventional molecule testing, RNA-sequencing and whole exon sequencing (WES) were performed as well to reveal underlying genetic landscape of this type of disease. Results: The median age of the 43 patients at diagnosis was 18.5 (range 4-114) months. The median while blood cell (WBC) counts and platelet at diagnosis were 17.2×10 9/L (range 1.8-105.7 ×10 9/L) and 36.5×10 9/L (range 4-472 ×10 9/L), respectively. Among them, 39.5% of cases exhibited hepatosplenomegaly. A diversity of genetic fusions and mutations were uncovered. The recurrent fusions in this cohort include CBFA2T3-GLIS2 (16.3%), MLLr (11.6%), NUP98-(9.3%), RBM15-MKL1 (7.0%), HOX fusions (7.0%) and PICALM-MLLT10 (4.7%). Other non-recurrent fusions accounted for 18.6% (Figure 1A). The common mutations detected in our pediatric AMKL were JAK2, NRAS, MPL, BCOR, CTCF, KRAS and PTPN11. Regarding treatment response, 20 of 43 cases (46.5%) attained complete remission (CR) or complete remission with incomplete blood cell recovery (CRi) after one cycle of induction. Among the 20 cases, 9 of them got minimal residual disease (MRD) negative by flow cytometry. Ten patients (23.2%) achieved partial remission (PR) while 13 patients (30.2%) showed no response (NR) to treatment. Thirty-one patients continued with another cycle of the same regimen and 21 of the 28 cases who were available for evaluation (75.0%) attained CR/CRi. Disease relapse was observed in 16/43 (37.2%) cases, with 7/43 (16.2%) patients relapsed after transplantation. The 3-year overall survival (OS) and event-free survival (EFS) were 49.6±8.2% and 29.5±7.1%, respectively (Figure 1B). In total, more than half of patients (22/43, 51.5%) underwent HSCT. The 3-year OS of transplant group and chemotherapy group were 57.9±11.7% versus 39.5±11.2%, P=0.036. As genetic characteristics often predict treatment response and prognosis, we wonder which subgroup could benefit from the reduced intensive induction. We further compared the features of patients who got CR/CRi after induction I with those achieved PR/NR. Interestingly, we found more patients with adverse fusions in PR/NR group, such as CBFA2T3-GLIS2 (26.0% vs. 5.0%), NUP98- (17.4% vs. 0%), MLLs (13.0% vs. 10.0%), and RBM15-MKL1 (8.7% vs. 5.0%); while other non-recurrent fusions were more common in CR/CRi group (4.3% vs. 35.0%). Two thirds of patients (67.4%) experienced refractory or relapse and almost half of them harbored adverse fusions or mutations such as CBFA2T3-GLIS2, MLLr, NUP98- fusions, NRAS and PTPN11. On the other hand, 14 patients without refractory or relapse had genetic features with more JAK2 and MPL mutations. No treatment-related mortality (TRM) occurred during induction. Only one patient with KRAS mutation died of infection after consolidation III. Conclusion: Insight into the genetic and molecular landscape improved our understanding of pediatric AMKL. Distinct molecular features can serve as a tool to tailor the therapy. Though treatment still lags behind and novel therapy is urgent for the adverse subgroups, our low-dose induction with HAG regimen could benefit for a subgroup with non-recurrent fusions, which showed efficiency while is tolerable for the young age group. Based on our results, we provide an induction option for these AMKL patients, especially for those in the middle-income countries.

Survival Outcomes of Acute Megakaryoblastic Leukemia in the United States: A 10-Year SEER-Based Study

Background: Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML), commonly observed in children with Down Syndrome (DS) but also seen in adults. The cytogenetic abnormality t(1;22)(p13.3;q13.1), has been reported in children with non-DS AMKL, which involves the fusion of RNA-binding motif protein-15 and megakaryoblastic leukemia-1 resulting in abnormal proliferation of megakaryocyte progenitors. Population-level data on outcomes of AMKL is scarce. Methods: We conducted a retrospective analysis using the Surveillance Epidemiology and End Results (SEER) database, a U.S. population-based registry maintained by the National Cancer Institute. We included all cases with histologically confirmed diagnoses of AMKL and AML with t(1;22)(p13.3;q13.1) between the years 2010 and 2019. Survival analysis was performed using the Kaplan-Meier method, and differences in overall survival (OS) were assessed using the log-rank test. Results: The analysis included a total of 217 cases, consisting of 54 patients with AML with t(1;22)(p13.3;q13.1) and 163 patients with AMKL. Among the AML cases with the cytogenetic abnormality, the median age was 58 years (ranging from 0 to 89 years), and 61% were female. 74% (n=40) of these patients received chemotherapy. The 1-year survival rate was 54.0% for the pediatric group (age < 18 years, n=10) and 44.6% for the adult group (age > 18 years, n=44) (Median OS 20 vs 8 months, p=0.69). Patients who received chemotherapy had a significantly higher 1-year survival rate of 57.9% compared to 14.3% in those who did not receive chemotherapy (p=0.0005). No significant difference in survival was observed when patients were grouped based on the year of diagnosis, with 1-year survival rates of 50.8% for the period 2010-2014 and 41.3% for 2015-2019 (p=0.66). Patients with AMKL were divided into two age groups: pediatric AMKL (age < 18 years, n=96) and adult AMKL (age > 18 years, n=67). The median age in this group was 3 years (ranging from 0 to 86 years), and 55% were male. 81% (n=132) of these patients were treated with chemotherapy. The 1-year survival rate for the pediatric group was 79.4%, whereas it was 17.6% for the adult group (p<0.0001). Chemotherapy significantly improved overall survival (OS) for both pediatric and adult groups, with 1-year survival rates of 82.5% vs. 35.7% in the pediatric group and 27.6% vs. 0.00% in the adult group in patients who did and did not receive chemotherapy, respectively (p=0.041 for pediatric group and p<0.0001 for adults). No significant differences in survival were observed among the groups based on the year of diagnosis, both in adults and the pediatric group. Conclusion: AMKL is commonly seen in infants and children, but it can affect individuals of all age groups. The prognosis is more favorable in pediatric patients. In contrast, outcomes for adults remain poor with current chemotherapeutic agents. Interestingly, AML with t(1;22)(p13.3;q13.1), which has been predominantly reported in children in the literature, was found to be more common in adults in this study. Regardless of age group, current treatments show poor outcomes. This study emphasizes on the urgent need for investigational agents.

Genomic Landscape of Pediatric Non-Down's Syndrome Acute Megakaryoblastic Leukemia in China

Acute megakaryoblastic leukemia (AMKL) accounts for 4% to 15% of newly diagnosed pediatric acute myeloid leukemia (AML), and different from Down's syndrome AMKL (DS-AMKL) and non-AMKL, clinical outcomes of non-DS-AMKL are characterized by an inferior prognosis. Although comprehensive genomic studies have revealed key genomic aberrations of non-DS-AMKL in Western cohort, there is still no reports about Chinese patients. Here we report a comprehensive transcriptomic study on 100 Chinese pediatric non-DS-AMKL cases through 2009 to 2022 in Shanghai Children's Medical Center. A total of 133 rearrangements were detected involving 78 patients, including previously reported recurrent fusion events which defining subgroups of AMKL, namely CBFA2T3-GLIS2 (n=20, 20%), NUP98-KDM5A (n=16,16%), rearrangements of HOX cluster genes (HOXr, n=11, 11%), RBM15-MKL1 (n=5, 5%) and MLLr (n=3, 3%). Two new recurrent gene translocations, XPO1-TRNC18 and STAG2 rearrangements (STAG2r) were detected in 5 (5%) and 11 (11%) cases, respectively. Further unsupervised clustering analysis was done with the most variant coding genes defined by standard deviation, the results showed that cases carrying STAG2r and XPO1-TNRC18 fusion exhibited distinguishable gene expression pattern from other subgroups and were clustered together respectively. It suggested these two rearrangements as potential driver genomic alterations in AMKL. By introducing XPO1-TNRC18 fusion and three STAG2 aberrations (STAG2 knock-out, STAG2-GNA12 and STAG2-OCRL) into 32D cell line, we proved these gene alterations promoted cell proliferation and colonies formation, suggesting potential function in tumor transformation. Based on these evidences, we defined XPO1-TNRC18 and STAG2r as two new subgroups of AMKL. We identified 222 nonsynonymous sequency mutations affecting 137 genes totally, including 202 single-nucleotide variants and 20 insertion/deletion, with a median of two mutations for per case (range 0-6). Among driver abnormalities, 7 aberrations occurred in more than 3% of cases in current cohort, including JAK2 (n=15, 15%), MPL (n=13, 13%), PTPN11 (n=7, 7%), NRAS (n=5, 5%), GATA1 (n=4, 4%), CTCF (n=4, 4%) and JAK3 (n=3, 3%). All the mutations could be clustered into 8 pathways, and the most frequently mutated pathways were JAK signaling (30%), transcription regulation (29%) and RAS signaling (14%). Mutation frequency varied across different subgroups. For example, JAK/STAT lesions were enriched in STAG2r subgroup (p<0.05). Distinct genomic aberration profile was revealed while comparing results of this non-DS-AMKL cohort to the mutation profile of non-AMKL cohort which we have published before. CBFA2T3-GLIS2, NUP98-KDM5A, STAG2r, XPO1-TNRC18 and RBM15-MKL1 subgroups were merely detected in AMKL, while driver aberrations like FLT3-ITD, CBFB-MYH1 and so on were not detected in AMKL. For driver mutations, JAK2 (15% vs 3.7%, p<0.0001), MPL (13% vs 0%, p<0.0001 ) and CTCF (4% vs 0%, p=0.007) were detected more frequently in non-DS-AML, while NRAS (5% vs 18.8%, p<0.001), KRAS (2% vs 10.6%, p=0.008), KIT (1% vs 21.6%, p<0.001), CSF3R (1% vs 6.1%, p=0.047), FLT3 (0% vs 22.5%, p<0.001), CEBPA (0% vs 9.8%, p<0.001), AXSL2 (0% vs 8.6%, p<0.001), GATA2 (0% vs 6.1%, p=0.007) and DHX15 (0% vs 4.5%, p=0.038) occurred less frequently in non-DS-AMKL cohort. Survival analysis revealed association between key aberrations and clinical outcomes. Carrying CBFA2T3-GLIS2 (p=0.018), NUP98-KDM5A (p<0.001) and FUS-ERG (p=0.018) was associated with unfavorable prognosis. Driver mutations in JAK2 (p=0.03) and RAS pathway (p=0.01) were associated with shorter overall survival. Among the two new subgroups, the new XPO1-TNRC18 subgroup demonstrated similar clinical outcomes to MLLr cases, with 5-year EFS and OS of 50%, and STAG2r was characterized by a favorable clinical outcome, with 5-year EFS and OS over 75%. As the first comprehensive genomic landscape of Chinese pediatric non-DS-AMKL, our results defined two new subgroups with distinct expression patterns and prognosis characteristics. We further revealed a distinct mutation profile between AMKL and non-AMKL and shed light on association between driver aberrations and clinical outcomes. These findings revealed non-DS-AMKL as a heterogenous disease, and suggested the importance of exploration of risk-stratification and targeted therapy in different mutations driven cases.

TRIP13 Is a Fetal-Enriched Therapeutic Target in NUP98-JARID1A + Pediatric Non-Down Syndrome AMKL

Chromosomal rearrangements have been identified as the main drivers of pediatric Acute Megakaryoblastic Leukemia in absence of Down syndrome (non-DS-AMKL). The t(11;12) involving NUP98 and KDM5A/JARID1A (NJ) accounts for approximately 15% of pediatric non-DS-AMKL cases and correlates with poor prognosis. An important aspect of AMKL is the strong enrichment in pediatric patients with frequent occurrence in infants (disease within 2 years post birth). In this study, we aimed to explore the role of cellular ontogeny in NJ-driven non-DS-AMKL and highlight TRIP13 as a fetal-enriched NJ-specific vulnerability acting through P53 regulation. To investigate the mechanism of NJ transformation, we first transduced murine hematopoietic stem and progenitor cells (HSPCs) originating from fetal liver (mFL-HSPCs) and bone marrow of adult mice (mBM-HSPCs) with a lentiviral vector overexpressing the cDNA encoding human NJ. In line with other studies, NJacts as a potent oncogene in vitro, sustaining cell proliferation and arresting cell differentiation. Both mFL-HSPCs and mBM-HSPCs expressing NJ transformed and dominated the culture after 3-4 weeks with an immature immune-phenotype (Lin-, Sca1+ and c-Kit+). Notably, outgrowth of mFL-HSPCs was significantly accelerated compared to adult mBM-HSPCs, despite similar transduction rates. Furthermore, in vivo experiments revealed that NJ-expressing mFL-HSPCs displayed a more aggressive phenotype (latency of 36 days) compared to mBM-HSPCs cells expressing NJ (latency of 70 days; P Long-rank < 0.001). Thus, both our in vitro and in vivo experiments strongly indicate an impact of fetal cell background. Suspecting an impact of fetal gene programs on NJ-mediated transformation, we performed a CRISPR-Cas9 screening targeting fetal expression signatures with a library probing 880 genes found to be deregulated when comparing fetal versus adult murine and human primary HSPCs. By comparing our findings with two other fetal liver-derived leukemia models - representing DS AMKL and familial platelet disorder with predisposition to AML (FPD-AML) -, we identified TRIP13 as a high-confidence candidate gene with exclusive dependency in NJ-driven non-DS-AMKL. Comparing human AML cell lines and NJ-overexpressing human fetal liver cells (hFL-HSPCs) confirmed selective targeting of NJ-driven cells by TRIP13 loss. To explore the molecular mechanism of TRIP13 sensitivity, we performed RNAseq. Surprisingly, gene set expression analysis (GSEA) revealed one highly significantly enriched pathway, i.e. TP53 signaling. Rescue experiments of Trip13 knockout in NJ-driven non-DS-AMKL cells further supported this finding: the massive depletion upon loss of Trip13 was completely reverted by either re-expression of a human TRIP13 cDNA or by knockout of Trp53. Of note, neither activation of TP53 signaling nor cell depletion was seen in healthy hematopoietic cells from FL upon Trip13 perturbation. Finally, we aimed to leverage TRIP13 dependency therapeutically. To this end, we first explored Trip13-depletion in an in vivo setting. In a fluorescence-based competitive transplantation assay, Trip13-ablated leukemic blasts were significantly diminished in the bone marrow of recipient mice after 4 weeks, showing a mean depletion of 83%. Next, NJ-driven non-DS-AMKL mFL-HSPCs were treated with the TRIP13 inhibitor DCZ0415. Similarly to genetic TRIP13 ablation, DCZ0415-treated NJ mFL-HSPCs showed a significantly higher sensitivity to the drug compared to models of DS AMKL, FPD-AML, and healthy HSPCs with fold changes of 1.6 (p-value = 0.024), 2.3 (p-value = 0.001) and 2.0 (p-value = 0.008), respectively. To exploit our mechanistic knowledge on TRIP13 ablation-mediated P53 activation, we further combined DCZ0415 with the MDM2 inhibitor Idasanutlin. In line with our mechanistic data, a high synergy (Bliss synergy score = 9.7) of TRIP13 and MDM2 inhibition was observed. In conclusion, our study uncovers TRIP13 as a fetal-enriched vulnerability in NJ-driven non-DS-AMKL, mechanistically acting through P53, which we leveraged for a mechanism-driven treatment approach with dual TRIP13/MDM2 inhibition as a potential therapeutic strategy for the treatment of high-risk pediatric non-DS-AMKL.

CBFA2T3::GLIS2 pediatric acute megakaryoblastic leukemia is sensitive to BCL-XL inhibition by navitoclax and DT2216

AbstractAcute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood–derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both invitro and invivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

Advances in molecular characterization of pediatric acute megakaryoblastic leukemia not associated with Down syndrome; impact on therapy development.

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia (AML) in which leukemic blasts have megakaryocytic features. AMKL makes up 4%-15% of newly diagnosed pediatric AML, typically affecting young children (less than 2years old). AMKL associated with Down syndrome (DS) shows GATA1 mutations and has a favorable prognosis. In contrast, AMKL in children without DS is often associated with recurrent and mutually exclusive chimeric fusion genes and has an unfavorable prognosis. This review mainly summarizes the unique features of pediatric non-DS AMKL and highlights the development of novel therapies for high-risk patients. Due to the rarity of pediatric AMKL, large-scale multi-center studies are needed to progress molecular characterization of this disease. Better disease models are also required to test leukemogenic mechanisms and emerging therapies.

High Fatality CBFA2T3-GLIS2 Pediatric Acute Megakaryoblastic Leukemia Is Sensitive to BCL-XL Inhibition

Acute megakaryoblastic leukemia (AMKL) is a rare subtype of pediatric acute myeloid leukemia (AML) with dismal survival prognosis in children below 3 years of age (<40% of cure rates). In two thirds of cases, recurrent and mutually exclusive oncogenic fusions are detected, such as CBFA2T3-GLIS2 (CG2) or NUP98 rearrangements (e.g. NUP98-KDM5A), which are considered the transformative event in this disease. The development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material, which is among other things attributed to myelofibrosis and the rarity of primary patients. Therefore, our research group has engineered synthetic human models of high-fatality pediatric AMKL with different oncogenic fusions that faithfully phenocopy the disease in a patho-physiological context. In recent years, inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, entered the therapeutic arena in adult AML and pediatric leukemia with promising results. The molecules emphasized on are Venetoclax (a specific inhibitor of BCL2) and Navitoclax (broader affinity to BCL2, BCL-XL and BCL-W). Even though we see promising results in other leukemia, these agents were so far not investigated as potential therapies in pediatric high-fatality AMKL. To first overcome the paucity of patient material for research, we generated synthetic models of high-risk AMKL by transducing cord-blood derived hematopoietic stem and progenitor cells (HSPC) with lentiviral particles carrying the CBFA2T3-GLIS2 oncogenic fusion, followed by xenotransplantation into NSG mice. This approach generated leukemia with a latency between 9.9 and 36.1 weeks in primary mice which are able to sustain serial rounds of transplantation in recipient mice (up to 5 rounds tested). Furthermore, leukemic blasts immunophenotypically recapitulate AMKL, demonstrating expression of megakaryopoietic markers such as CD41, CD61 and CD56 that are even maintained after in vitro culture of 6 days and subsequent transplantation. Furthermore, we derived a CG2 specific gene expression signature from transcriptomic profiling of our models as well as patient samples that showed high expression of NCAM1, BMP2, ERG and low GATA1 levels, as reported for CG2 leukemia. Remarkedly, one of the specific up regulated genes in our signature for CG2 was pro-survival factor BCL2. In addition, our models are located in the megakaryocytic-erythroid differentiation space, as assessed by single cell RNAseq, and similar to their healthy counterparts of the megakaryopoietic lineage they demonstrated high expression of BCL-XL on RNA and protein level, in contrast to AML samples. Based on the aberrant expression of BCL2 in our synthetic leukemia models and lineage related expression of BCL-XL, we set out to test Venetoclax and Navitoclax in the context of AMKL. We identified Navitoclax (inhibitor of BCL2, BCL-XL and BCL-W) as a therapeutic vulnerability in our models of AMKL (CG2 and NUP98 rearranged), whereas our cells demonstrated resistance to treatment with Venetoclax. We further investigated the molecular mechanism and showed that shRNA mediated knock-down or inhibition of BCL-XL, by either Navitoclax or the BCL-XL specific proteasomal degrader DT2216, results in significant induction of apoptosis in our models of AMKL. On the contrary, genetic or pharmacological inhibition of BCL2 did not induce apoptosis in our synthetic models of AMKL, even at doses as high as 10µM. Furthermore, a genotype matched leukemic model of NUP98-KDM5A, which was presenting as monocytic (CD68+LYZ+) AML instead of AMKL, was resistant to either treatment with Venetoclax or Navitoclax whereas ML2, a MLL rearranged AML cell line, showed increased sensitivity to both Venetoclax and Navitoclax treatment. Moreover, in vivo treatment of a xenografted CG2 AMKL model with Navitoclax reduced leukemic burden in mice in bone-marrow and spleen in comparison to vehicle treated controls. In conclusion, we generated a model of rare CG2 pediatric high-fatality leukemia that faithfully mimics the patient situation and allows biomass generation for large scale multi-omic approaches. Furthermore, we demonstrate a lineage- and genotype specific targetable dependency of pediatric AMKL towards inhibition of BCL-XL but not BCL2, that could be transferred to the clinic as a novel therapeutic option in high-risk infant leukemia.

Pediatric non-Down's syndrome acute megakaryoblastic leukemia patients in China: A single center's real-world analysis.

Non-Down’s syndrome acute megakaryocytic leukemia (non-DS-AMKL) is a subtype of childhood acute myeloid leukemia (AML), whose prognosis, prognostic factors and treatment recommendations have not yet to be defined in children. We conducted a retrospective study with 65 newly diagnosed non-DS-AMKL children from August 2003 to June 2020 to investigate the clinical impact of factors and clinical outcome. Among all 65 patients, 47 of them were treated at our center who received three different regimens due to time point of admission (CAMS-another, CAMS-2009 and CAMS-2016 protocol), and the efficacy were compared. Patients with newly diagnosed non-DS-AMKL accounted for 7.4% of pediatric AML cases. The median age of the patients was 18 months at diagnosis, and over 90% of them were under three-years-old. The overall survival (OS) rates were 33.3% ± 1.7%, 66.7% ± 24.4% and 74.2% ± 4.0% for three groups (CAMS-another, CAMS-2009 and CAMS-2016 regimen), respectively. In CAMS-2016 group, the complete remission (CR) rate after induction was 67.7% (21/31), while the total CR rate after all phases of chemotherapy was 80.6% (25/31). The 2-year survival probability did not significantly improve in patients underwent HSCT when compared with non-HSCT group (75.0% ± 4.7% vs. 73.9% ± 4.6%, p=0.680). Those who had a “dry tap” during BM aspiration at admission had significantly worse OS than those without “dry tap” (33.3% ± 8.6% vs. 84.0% ± 3.6%, p=0.006). Moreover, the results also revealed that patients with CD34+ had significantly lower OS (50.0% ± 6.7% vs. 89.5% ± 3.5%, p=0.021), whereas patients with CD36+ had significantly higher OS than those who were negative (85.0% ± 4.0% vs. 54.5% ± 6.6%, p=0.048). In conclusion, intensive chemotherapy resulted in improved prognosis of non-DS-AMKL children and subclassification may base on “dry tap” and immunophenotypic. Although some progress has been made, outcomes of non-DS-AMKL children remain unsatisfactory, especially in HSCT group, when compared with other AML types.

CLINICAL AND MOLECULAR REPORT OF A RARE RAM POSITIVE NON-DOWN SYNDROME INFANT CBFA2T3-GLIS2 NEGATIVE, PRESENTING THE DRIVER P210-BCR-ABL1

Pediatric Acute Megakaryoblastic Leukemia (AMKL) comprises 4%‒15% of Acute Myeloid Leukemia (AML) cases. Down Syndrome (DS)-AMKL usually displays a favorable outcome, whereas the prognosis of non-DS-AMKL is generally poor. Here we present the clinical and genomic data of a rare non-DS-AMKL infant with p210-BCR-ABL1 fusion, co-occurring with a high-risk WT1 mutation. The baby was RAM positive and CBFA2T3-GLIS2 negative, showing refractoriness to treatment. A 1-year-old girl was referred to the Hospital with a history of fever and motor developmental delay for four months. On admission, she presented ecchymosis, swelling, and organomegaly (spleen and liver). The WBC count was 26.5 × 10 3 /μL, hemoglobin level was 7.6 g/dL, platelet count was 25 × 10 3 /μL, and LDH was 2.887. At diagnosis, she showed bilateral myeloid sarcoma. Morphological analysis of the Bone Marrow (BM) showed clustered cytoplasmic blebs. Flow cytometry analysis revealed 83% of blast cells and a profile compatible with AMKL and RAM phenotype. The patient was treated under the high-risk AML-BFM-2004. Imatinib was incorporated into the treatment after BCR-ABL1 detection. However, minimal residual disease remained positive throughout the treatment, making BM transplantation unfeasible. Disease refractoriness was persistent even with the administration of Dasatinib, FLAG-IDA and Venetoclax. The patient died one year after diagnosis due to sepsis during BM aplasia. We performed G-banding, FISH, and Multiplex RT-PCR experiments. The molecular response was assessed by absolute quantification via RT-qPCR assays. Sanger sequencing and NGS were performed on a targeted approach. The karyotype was 46,XX,t(9;22)(q21;q34). We observed polyploidy (3‒20N). FISH in polyploid Nuclei (8N) showed four BCR-ABL1 fusion signals. In diploid metaphases, we observed two BCR-ABL1 signals. Multiplex RT-PCR detected the p210- BCR - ABL1 . RT-qPCR monitoring detected persistent high levels of BCR-ABL1 transcripts associated with disease refractoriness. Sanger sequencing showed no GATA1 mutation. NGS revealed a missense WT1 pathogenic mutation, and CBFA2T3-GLIS2 was not present. RAM phenotype is characterized by overexpression of CD56 and under-expression of CD38, CD45, and HLA-DR. This phenotype is characteristic of children, usually associated with AML-M7 (38%) and intermediate-risk cytogenetics not associated with prognostic subgroups. RAM is related to a poor prognosis with high MRD and induction failure rates compared to non-RAM patients. This phenotype is associated with the CBFA2T3-GLIS2 fusion, which is related to extramedullary involvement, a particular blast morphology, and a worse outcome as compared to fusion-negative AML pediatric patients, including a high frequency of disease refractoriness and relapse. NGS studies did not show CBFA2T3-GLIS2 in our patient. Thus, our results indicate that RAM+ associated with the driver p210-BCR-ABL1, even with specific targeted therapy, may lead to treatment resistance regardless of CBFA2T3-GLIS2+. Here we highlight for the first time the presence of p210-BCR-ABL1 associated with RAM phenotype in the absence of CBFA2T3-GLIS2 and disease refractoriness in a non-DS-AMKL infant.

Revealing the intratumoral heterogeneity of non-DS acute megakaryoblastic leukemia in single-cell resolution.

Pediatric acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) characterized by abnormal megakaryoblasts, and it is divided into the AMKL patients with Down syndrome (DS-AMKL) and AMKL patients without DS (non-DS-AMKL). Pediatric non-DS-AMKL is a heterogeneous disease with extremely poor outcome. We performed single-cell RNA sequencing (scRNA-seq) of the bone marrow from two CBFA2T3-GLIS2 fusion-positive and one RBM15-MKL1 fusion-positive non-DS-AMKL children. Meanwhile, we downloaded the scRNA-seq data of normal megakaryocyte (MK) cells of the fetal liver and bone marrow from healthy donors as normal controls. We conducted cell clustering, cell-type identification, inferCNV analysis, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Monocle2 analysis to investigate the intratumoral heterogeneity of AMKL. Using canonical markers, we identified and characterized the abnormal blasts and other normal immune cells from three AMKL samples. We found intratumoral heterogeneity of AMKL in various cell-type proportions, malignant cells’ diverse copy number variations (CNVs), maturities, significant genes expressions, and enriched pathways. We also identified potential markers for pediatric AMKL, namely, RACK1, ELOB, TRIR, NOP53, SELENOH, and CD81. Our work offered insight into the heterogeneity of pediatric acute megakaryoblastic leukemia and established the single-cell transcriptomic landscape of AMKL for the first time.

3173 – HUMAN MODELS OF PEDIATRIC CBFA2T3-GLIS2 ACUTE MEGAKARYOBLASTIC LEUKEMIA CONTRIBUTE TO UNCOVER THERAPEUTIC VULNERABILITIES

Acute megakaryoblastic leukemia (AMKL) is a poor prognostic subtype of AML afflicting children mostly under 3 years of age. Chromosomal translocations generating chimeric fusion genes are considered critical transforming events and are present in a high proportion of cases. Among them, the CBFA2T3-GLIS2 (CG2) fusion is associated with a high risk of relapse and accounts for ∼15% of pediatric AMKL cases. Using overexpression of CG2 in human cord blood CD34+ stem /progenitor cells (hHSPC) followed by xenotransplantation in immunodeficient mice, we are reporting the generation of multiple synthetic AMKL models that phenocopy the human disease in term of immunophenotype, morphology and gene expression. CG2 models revealed a stable genomic landscape with few copy number anomalies and lack of recurrent cooperating mutations. Leveraging from local and published transcriptomic datasets of CG2 AMKL samples (14 patients, 10 models), non-CG2 pediatric AMKL (n=66), NUP98-KDM5A synthetic models (n=5) and normal hHSPC (n=4), we have determined a robust gene expression signature defining CG2 AMKL. Gene set enrichment analysis of CG2 AMKL expression profile identified the JAK2-STAT5 axis as a potential pathway that is upregulated in CG2 leukemia, confirmed by downstream STAT5 phosphorylation in CG2 cells. Cell surface proteomic analysis by LC-MS identified multiple membrane proteins upstream of JAK-STAT signaling in CG2 model leukemia compared to normal hHSPC. Single-agent dose-response curves conducted with multiple FDA-approved drugs targeting JAK2 showed specific grow inhibition of CG2 AMKL models compared to normal hHSPC and non-AMKL cell line. Progress in leukemia research is hampered by the paucity of patient samples and the generated AMKL models pave the way toward a better understanding of leukemogenesis and development of targeted therapy. Acute megakaryoblastic leukemia (AMKL) is a poor prognostic subtype of AML afflicting children mostly under 3 years of age. Chromosomal translocations generating chimeric fusion genes are considered critical transforming events and are present in a high proportion of cases. Among them, the CBFA2T3-GLIS2 (CG2) fusion is associated with a high risk of relapse and accounts for ∼15% of pediatric AMKL cases. Using overexpression of CG2 in human cord blood CD34+ stem /progenitor cells (hHSPC) followed by xenotransplantation in immunodeficient mice, we are reporting the generation of multiple synthetic AMKL models that phenocopy the human disease in term of immunophenotype, morphology and gene expression. CG2 models revealed a stable genomic landscape with few copy number anomalies and lack of recurrent cooperating mutations. Leveraging from local and published transcriptomic datasets of CG2 AMKL samples (14 patients, 10 models), non-CG2 pediatric AMKL (n=66), NUP98-KDM5A synthetic models (n=5) and normal hHSPC (n=4), we have determined a robust gene expression signature defining CG2 AMKL. Gene set enrichment analysis of CG2 AMKL expression profile identified the JAK2-STAT5 axis as a potential pathway that is upregulated in CG2 leukemia, confirmed by downstream STAT5 phosphorylation in CG2 cells. Cell surface proteomic analysis by LC-MS identified multiple membrane proteins upstream of JAK-STAT signaling in CG2 model leukemia compared to normal hHSPC. Single-agent dose-response curves conducted with multiple FDA-approved drugs targeting JAK2 showed specific grow inhibition of CG2 AMKL models compared to normal hHSPC and non-AMKL cell line. Progress in leukemia research is hampered by the paucity of patient samples and the generated AMKL models pave the way toward a better understanding of leukemogenesis and development of targeted therapy.

3093 – SUBTYPE-SPECIFIC INDUCTION OF MITOCHONDRIAL APOPTOSIS AS A NOVEL THERAPEUTIC OPTION IN HIGH-FATALITY PEDIATRIC LEUKEMIA

A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML. A rare subtype of acute myeloid leukemia (AML) is acute megakaryoblastic leukemia (AMKL). Recurrent and mutually exclusive oncogenic fusions are detected in AMKL and are considered the transforming event in this disease. AMKL usually affects children below 3 years of age and is associated with less than 40% cure rates. In addition, the development of genotype tailored therapies is urgently needed in AMKL but greatly limited by the paucity of primary sample material. Our research group has established synthetic human models of high-fatality pediatric AMKL, driven by distinct oncogenic fusions, which phenocopy the disease in a patho-physiological context in mice. Inducers of mitochondria-mediated apoptosis, so called BH3 mimetics, recently entered the therapeutic arena in pediatric leukemia with promising results. Therefore, we set out to investigate the potential of Venetoclax (inhibitor of BCL-2) and Navitoclax (inhibitor of BCL-2, BCL-XL and BCL-W) as novel therapeutic options as a bridge to curative bone marrow transplantation in this high-fatality infant leukemia. Our research demonstrates that genetic and pharmacological inhibition of the pro-survival BCL-2 family member BCL-XL results in significant induction of apoptosis in our models of AMKL. In contrast, inhibition of BCL-2 does not induce apoptosis in our models, which is currently the most investigated BCL-family member in the clinic for other subtypes of AML (Venetoclax). In addition, Navitoclax showed promising in vivo activity with a significant decrease in infiltrating leukemic cells in bone-marrow and spleen after drug treatment in comparison to vehicle-only controls. In summary, this project will bring mechanistic insight into the role of apoptotic pathways in AMKL and greatly accelerate the identification of novel therapeutic options in high-fatality infant AML.

The Role of ETO and CtBP1 in CBFA2T3-GLIS2 Mediated Transcriptional Regulation and Leukemogenesis

CBFA2T3-GLIS2 is the most prevalent fusion oncogene in pediatric acute megakaryoblastic leukemia patients without Down syndrome and is associated with an event free survival of only 8%. A cryptic inversion event on chromosome 16 joins the three nervy homology regions (NHR) of CBFA2T3 to the five zinc fingers of GLIS2. This configuration enables the encoded chimeric transcription factor to bind GLIS2 consensus sequences throughout the genome and recruit transcriptional activators and repressors to alter gene expression and enhance self-renewal capability. Few cooperating mutations have been identified in patients harboring this fusion which suggests it is the sole oncogenic driver. The molecular mechanism by which CBFA2T3-GLIS2 drives leukemogenesis is not fully understood. Identification of components critical to the transcriptional complex and their role in gene regulation may reveal novel therapeutic targets to improve patient outcomes. Studies on the wild type CBFA2T3 and GLIS2 proteins have demonstrated interactions with the transcriptional regulators ETO and CtBP1 respectively. Further p300 has been shown to play a role in transcriptional regulation imparted by both transcription factors. We therefore hypothesize the fusion promotes transcriptional activation when the histone acetyltransferase p300 and the transcription factor ETO are recruited through NHR1 and NHR2, respectively. When the co-repressor CtBP1 is recruited through the PXDLS motif, located in the GLIS2 portion of the fusion, transcriptional repression predominates. Association of these co-factors with the fusion was confirmed through co-immunoprecipitations. Site-directed mutagenesis was then used to systematically delete NHR1 and NHR2 and mutate the PXDLS motif to evaluate the resultant effects on transcriptional regulation, self-renewal, and leukemogenesis imparted by the fusion. A luciferase reporter assay was used to assess transcriptional activation of the BMP2 promoter, a gene which is known to be upregulated by the CBFA2T3-GLIS2 fusion. Loss of NHR1, NHR2, or NHR3 did not alter the ability of the fusion to activate transcription. In contrast, loss of NHR1 and NHR2 in combination (NHR1-2Δ) and mutation of the PXDLS domain decreased transcriptional activation compared to the wild type fusion. The effect of the mutations on self-renewal capability was then evaluated through colony formation assays. Consistent with the luciferase reporter assay, NHR1-2Δ and PXDLS mutants decreased the number of colonies at week six compared to the unmanipulated fusion. Next, we investigated the effect of these mutations on leukemogenesis. Murine models harboring the CBFA2T3-GLIS2 fusion without cooperating mutations have been unsuccessful and patient-derived xenograft models are limited and difficult to manipulate. Therefore, we developed a novel in vivo model of CBFA2T3-GLIS2 driven leukemia. CD34+ stem cells were isolated from human cord blood and transduced with a lentivirus construct encoding the fusion and a GFP reporter. The cells were then differentiated to megakaryoblasts using human TPO and IL1-beta and sorted for purity prior to injection into immunodeficient NSG-SGM3 recipient mice. The fusion positive human primary megakaryoblasts induced a serially transplantable leukemia within 180 days that recapitulates CBFA2T3-GLIS2 positive patient specimens on a transcriptional and protein level. In contrast to our in vitro studies where NHR2 deletion alone did not alter transcriptional activation and self-renewal, the loss of this domain abrogated leukemogenesis in vivo, suggesting a dependency on the association of ETO with the fusion. Mice that received PXDLS mutant cells, however, developed leukemia at a normal latency suggesting that CtBP1 is not required. This study confirms the CBFA2T3-GLIS2 fusion is sufficient for oncogenic transformation of human CD34+ stem cells. We demonstrate that disruption of ETO, p300, and CtBP1 recruitment to the transcriptional complex decreases transcriptional regulation and self-renewal imparted by the fusion. The loss of ETO was the most detrimental to leukemogenesis in our murine model, uncovering a potential new pathway for the development of targeted therapies. Ongoing studies include CUT&RUN sequencing for the fusion, ETO, and CtBP1 to determine co-occupancy of target genes to further understand those that are critical in transformation. DisclosuresGruber: Kura Oncology: Consultancy.

Epigenetically Enhanced MED12L in ETO2-GLIS2 Positive Pediatric Acute Megakaryoblastic Leukemia Is Associated with Resistance to the CDK8 Inhibitors

Introduction. ETO2-GLIS2 (aka CBFA2T3-GLIS2) is the most common (30%) alteration in pediatric de novo acute megakaryoblastic leukemia (AMKL). These patients have poor response to induction therapy, a high incidence of relapse (~90%), and dismal 5-year survival rates (<20%). Previous studies suggest that ETO2-GLIS2 induces leukemia through abnormal enhancer formation as a single oncogenic “hit”. Because ETO2-GLIS2 expression induces formation of leukemia-specific neo-superenhancer (SE) elements, we hypothesize that mediator (MED) proteins are involved in linking neo-SE elements to distal gene expression, and thus could be a therapeutic target. Here, we analyzed expression of MED-family genes in ETO2-GLIS2 positive AMKL patients in combination with enhancer-histone marks, and evaluated impact of MED-kinase inhibition with selective CDK8 inhibitors (CDKi) against cell viability.Methods. To address our hypothesis, we analyzed published RNA sequencing dataset (Smith et al. 2020) for human MED-genes expression from a pan-pediatric AML cohort (N=1476). The cohort consisted of subgroups expressing fusions of ETO2-GLIS2 (N=40), CBFB-MYH11 (N=174), DEK-NUP214 (N=49), KMT2A-ELL (N=50), KMT2A-MLLT10 (N=86), KMT2A-MLLT3 (N=114), KMT2A-MLLT4 (N=49), NUP98-NSD1 (N=107), RUNX1-RUNX1T1 (N=210) and no detectable fusions (N=526), compared to normal bone marrow (NBM) samples (N=71). Enrichment of histone marks overlapping MED-genes was analyzed from published chromatin immunoprecipitation (ChIP) sequencing in ETO2-GLIS2 positive M-07e AMKL line (Thirant et al. 2017). We tested efficacy of CDK8 inhibition with BI-1347 and CCT251545 against M-07e cells to determine their activity in the context of marked MED12L overexpression.Results. We examined expression of 29 MED-genes comprising the 4 major (head, middle, tail, and kinase) MED-modules. MED gene expression was variable across AML subtypes and NBM. However, MED genes were more commonly over-expressed in the ETO2-GLIS2 group, in particular MED 17, MED1, MED10, MED27, and MED12L (paralog of MED12) were upregulated in the subgroup. Most notably, we noted exceptional upregulation of MED12L (FC 4.9, log2), compared to NBM (Figure 1). Because MED12/12L plays an intrinsic biological role in establishing oncogenic enhancer-expression loops in hematopoietic stem or leukemic cells, we investigated the overlap of enhancer bound histones such as H3K27ac and H3K4me1 to MED12L in M-07e cells, compared to umbilical cord blood-derived normal megakaryoblasts (MK) (S004BT; Blueprint epigenome database). We found enrichment of H3K4me1 at MED12L transcription start site (TSS) and upstream promoter both in MK and M-07e cells. In addition, we observed a large region of H3K27ac enrichment spanning 89 Kb (16 kb upstream and 73 kb downstream) across MED12L TSS in M-07e cells, suggesting neo-enhancer activity at this locus. Considering the dependency of MED12 on CDK8 for MED-kinase activities (Klatt et al. 2020), we treated M-07e cells with CDK8i(s), to test our hypothesis if perturbation of epigenetically enhanced MED12L expression can impact leukemic growth. However, we observed a poor correlation between M-07e cell viability and IC 50 of BI-1347 (IC 50: 0.87 µM, R 2: 0.36) or CCT251545 (IC 50: 0.4 µM, R 2: 0.48). In contrast, ETO2-GLIS2 negative MV4-11 AML cells were susceptible to both BI-1347 (IC 50: 0.44 µM, R 2: 0.88) and CCT251545 (IC 50: 0.12 µM, R 2: 0.87). Given the inefficacy of CDK8i against M-07e, and cooperativity of bromodomain extra-terminal (BET)-BRD4 and MED12/12L in forming enhancer complexes, we tested possible inhibitory impact of BRD4 inhibitor JQ1 on MED12L expression and leukemic growth in target cells. We observed effective reduction in M-07e cell viability (IC 50:0.3 µM, R 2: 0.93) with concomitant reduction not only in BRD4 protein-expression, but diminished MED12L protein expression at IC 50 and higher doses of JQ1.Conclusion. Our findings revealed that MED12L is highly overexpressed and overlapped with strong neo-enhancer chromatin-marks in ETO2-GLIS2 positive cells, while maintaining resistance to CDK8i. Future studies will contribute to deeper insights into the preferential recruitment and role of MED12L in ETO2-GLIS2 bound enhancers and potential mechanisms of resistance to CDK8 inhibition in the disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transcriptomic and Network Signatures of Cryptic Fusion CBFA2T3-GLIS2 in Pediatric AML Reveals Clinical Considerations and Implications

IntroductionA cryptic inv(16)(p13.3q24.3) encoding the CBFA2T3-GLIS2 fusion was initially reported in childhood acute megakaryoblastic leukemia (AMKL) and was shown to be associated with unfavorable outcomes. Although this fusion onco-protein is more prevalent in FAB M7 AML, it is reported in younger patients with all subtypes of AML. We present a comprehensive transcriptomic signature of an unselected cohort of children with CBFA2T3-GLIS2 fusion treated on COG AML trials.MethodsCBFA2T3-GLIS2 fusions were identified in 26 of 1042 (2.5%) patients enrolled in Children's Oncology Group (COG) studies AAML03P1 and AAML0531, with a prevalence of 8% in patients < 3 years of age. Whole transcriptome sequence data was available from 466 patients and 20 normal bone marrows (NBM) for expression profiling. Differentially expressed genes were identified using the Limma v.3.30.12 R package. Unsupervised hierarchal clustering was performed using Euclidean distance with the WPGMA linkage algorithm. Gene-set enrichment analysis was completed using the Broad Institute software v.2-2.2.4 with MSigDB C2 curated gene sets.ResultsOf 26 patients with CBFA2T3-GLIS2 fusions, 21 (81%) were < 5 years of age (Fig. 1A). KMT2A fusions co-occurred in only two patients, and none of the positive tumors carried core binding factor fusions. Few other variants accompanied CBFA2T3-GLIS2 fusions and overall, this subclass of AML had been categorized as cytogenetically normal (42% CN-AML, Fig. 1B). CBFA2T3-GLIS2 AML fared worse than the AML population as a whole; more than 75% of fusion-positive cases relapsed within two years (Fig. 1C, p = 0.001).Gene-set enrichment analysis between CBFA2T3-GLIS2 fusion and fusion-negative tumors revealed the top three positively enriched gene sets as 1) basal cell carcinoma (FDR = 0.003), 2) ECM receptor interactions (FDR = 0.006), and 3) hedgehog signaling pathways (FDR = 0.029). Consistent with previous reports, genes associated with WNT, hedgehog (HH), and BMP signaling were significantly up-regulated in CBFA2T3-GLIS2 tumors (Fig. 1D). CBFA2T3-GLIS2 fusions have been associated with induction of BMP signaling through downstream HH targets, BMP2 and BMP4 in pediatric AMKL. Moreover, concurrent dysregulation of WNT, BMP and HH signaling is a feature of CBFA2T3-GLIS2 AMKL (Gruber et al., 2015). Unsupervised hierarchical clustering of differentially expressed genes effectively clustered the fusion-positive samples, suggesting CBFA2T3-GLIS2 fusions drive a distinct transcriptional profile from other AML subtypes (Fig. 1E). CBFA2T3-GLIS2 tumors compared to NBM had upregulated expression of 4 cancer testes antigens (CTAs): CCNA1, PRAME, IGF2BP3, and KDM5B (fold-change > 2, adj. p < 0.001). CTAs are promising candidates for targeted T-cell therapies due their low expression in NBM and surrounding tissues, which could limit treatment related toxicity ( Ochsenreither et al., 2012) .We further performed unsupervised hierarchical clustering in 437 patients for whom flow cytometry data were available (Fig. 1F). Most fusion-positive tumors (7/10) clustered together by mean fluorescence intensity (MFI), indicating that CBFA2T3-GLIS2 tumors have highly distinctive immune profiles. Of particular interest, fusion-positive tumors expressed CD56 at high levels (median CD56 in CBFA2T3-GLIS2 : 1,505.0 MFI [range: 6.4 - 4,290.0] versus median CD56 in fusion-negative AML: 9.0 MFI [range: 2.4 - 3,360.0], p = 0.001). A novel immunophenotype in pediatric AML, termed RAM, has been associated with high CD56 expression and much lower event-free survival. A preponderance of CBFA2T3-GLIS2 AML displayed the RAM phenotype, with 70% of fusion-positive tumors identified as RAM compared to 0.23% (1/427) in the fusion-negative population (p < 0.001).ConclusionCBFA2T3-GLIS2 AML tends to have an aggressive disease pathology in much younger patients, who had been primarily classified as CN-AML. This suggests screening for CBFA2T3-GLIS2 in younger patients could be valuable as standard practice, though not yet recommended by the World Health Organization. Our associations with CBFA2T3-GLIS2 fusions provides a potential link between genomic and immunologic characteristics of a high-risk group of pediatric AMLs, which could be employed in the future for clinical screening through molecular or flow-cytometric methods. [Display omitted] DisclosuresBrodersen:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership.

The ETO2-GLIS2 Fusion Oncogene Alters Early Human Hematopoiesis in an Induced Pluripotent Stem Cells-Derived Model of Pediatric Acute Megakaryoblastic Leukemia

De novo pediatric acute megakaryoblastic leukemia (AMKL) is a genetically heterogeneous subtype of acute myeloid leukemia (AML) expressing fusion oncogenes, including CBFA2T3 - GLIS2 (a.k.a. ETO2-GLIS2) present in 15-25% of patients and associated with a poor prognosis because of a high risk of relapse or refractoriness to chemotherapy. To characterize the consequences of ETO2-GLIS2 on early stages of human hematopoiesis, we developed a model of ETO2-GLIS2 expression in induced pluripotent stem cells (iPSC) derived from healthy human CD34+ cells. To this end, we introduced the human ETO2-GLIS2 cDNA tagged with the green fluorescent protein (GFP) under the control of the human CD43 promoter in iPSC through ZFN-mediated recombination at the AAVS1 safe-harbor locus. After puromycin selection, 23 independent clones were confirmed by PCR and sequencing to bear a correct homozygous integration and were amplified. Three independent iPSC clones were subsequently differentiated into hematopoietic progenitors and then into megakaryocytes using a 2D co-culture system on matrix adapted from Chou S et al. (PNAS 2012) and compared to control iPSC. We demonstrated that ETO2-GLIS2 is expressed in CD43+ hematopoietic cells both at the transcript and protein levels and localizes in the nucleus. In addition, ETO2-GLIS2-expressing cells showed an increased expression of several recently characterized direct targets of the fusion including ERG as compared to control cells. Hematopoietic differentiation of iPSC was then assessed at day 13, 15 and 18 using flow cytometry analyses for progenitors and megakaryocytic markers. As early as day 13, ETO2-GLIS2-expressing cells showed an increased number of CD43+ hematopoietic cells composed of a higher percentage of CD41+CD42+ megakaryocytic cells compared to controls, indicating that ETO2-GLIS2 enhances early human megakaryocyte development. Importantly, we observed strong differentiation alterations, including a lower expression of CD61 on ETO2-GLIS2 megakaryocytes compared to controls at all time points and a progressive accumulation of an abnormal CD41lowCD42+ population never detected in control conditions. This CD41lowCD42+ population was also observed in AMKL patients. Finally, preliminary clonogenic assays in methylcellulose indicate that ETO2-GLIS2 enhances serial replating activity for several weeks. These data support the idea that expression of ETO2-GLIS2 in human hematopoietic cells obtained from iPSC recapitulates several cellular and molecular features of human pediatric AMKL, including transcriptional activation of target genes, enhanced self-renewal of progenitors and aberrant differentiation of megakaryocytic cells. This model will facilitate further cellular and preclinical studies on this rare pediatric cancer. DisclosuresNo relevant conflicts of interest to declare.

Outcome and Prognostic Features in Pediatric Acute Megakaryoblastic Leukemia Without Down Syndrome: A Retrospective Study in China

BackgroundAcute megakaryoblastic leukemia (AMKL) is a biologically heterogeneous subtype of acute myeloid leukemia that originates from megakaryocytes. Patients with AMKL with non-Down syndrome (DS) had a poorer prognosis. However, clear prognostic indicators and treatment recommendations for this subgroup remain controversial. Patients and MethodsHerein, we performed a retrospective study on 40 patients (age ≤ 18 years) with non-Down syndrome AMKL at our institution. We assessed the effect of different prognostic factors, such as their cytogenetic abnormalities, early treatment response, and the role of hematopoietic stem cell transplantation (HSCT) as post-remission treatment on the outcomes. ResultsThe complete remission (CR) rate of the patients was 57.9% and 81.1%, respectively, at the end of induction therapy 1 and 2. The overall survival (OS) and event-free survival rates at 2 years were 41% ± 13% and 41% ± 10%, respectively. An analysis of the cytogenetic features showed that patients with +21 or hyperdiploid (> 50 chromosomes) had significantly better OS than those in other cytogenetic subgroups (Plog-rank = .048 and Plog-rank = .040, respectively). Besides cytogenetics, an excellent early treatment response (CR and minimal residual disease < 1% after induction therapy 1) also provided a significant survival benefit in univariate analysis in our study. However, multivariate analysis indicated that allogeneic HSCT was the only independent prognostic marker (relative risk, 11.192; 95% confidence interval, 2.045-61.241; P = .005 for OS and relative risk, 5.400; 95% confidence interval, 1.635-17.832; P = .006 for event-free survival, respectively). ConclusionAMKL in patients with non-Down syndrome has a poor outcome. With poor OS but CR rates comparable with other acute myeloid leukemia subtypes, allogenic HSCT may be a better option for post-remission therapy than conventional chemotherapy, especially for those having a poor response to induction therapy.