Regulatory Networks of Prognostic mRNAs in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) in children refers to a malignant tumor caused by the abnormal proliferation of immature myeloid cells in the bone marrow and peripheral blood. The prognosis of patients with pediatric acute myeloid leukemia (AML) remains poor, highlighting the need for improved targeted therapy. The expression data of lncRNAs, mRNAs, and miRNAs and survival information of pediatric AML patients were collected from The Cancer Genome Atlas (TCGA) database. Cox regression analysis was used to screen the lncRNAs, mRNAs, and miRNAs that significantly affect the overall survival (OS) of patients as OS-related genes (included lncRNAs, mRNAs, and miRNAs). Enrichment analysis and protein-protein interaction (PPI) network construction were performed for the OS-related mRNAs. We further established a ceRNAs regulatory network. In addition, the potential prognostic role of genes was further evaluated by risk score. We have identified 5275 lncRNAs, 176 miRNAs, and 6221 mRNAs that significantly affect the prognosis of pediatric AML patients. It is worth noting that OS-related mRNAs are mainly involved in ribosome, RNA transport, and spliceosome. We identified the top 10 most connected mRNAs in the PPI network as important mRNAs and constructed a ceRNAs regulatory network (including NCBP2, RPLP0, UBC, RPS2, and RPS9). The risk score and nomogram results suggest that NCBP2 may be a risk factor for pediatric AML, while RPLP0, UBC, RPS2, and RPS9 may be protective factors. Our results construct 5 gene signals as new prognostic indicators for predicting the survival of pediatric AML patients. Our research has demonstrated the ceRNAs regulatory network may become a new target for pediatric AML treatment.

Integrated Genomic Analysis Identifies UBTF Tandem Duplications As a Subtype-Defining Lesion in Pediatric Acute Myeloid Leukemia

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

Immunotherapeutic Targeting of Mesothelin Positive Pediatric AML Using Bispecific T Cell Engaging Antibodies

Advances in the treatment of pediatric AML have been modest over the past four decades. Despite maximally intensive therapy, approximately 40% of patients will relapse. Novel targeted therapies are needed to improve outcomes. We identified mesothelin (MSLN), a well-validated target overexpressed in some adult malignancies, to be highly expressed on the leukemic cell surface in a subset of pediatric AML patients. The lack of expression on normal bone marrow cells makes MSLN a viable target for immunotherapies such as T-cell engaging bispecific antibodies (BsAbs) that combine two distinct antibody-variable regions into a single molecule targeting a cancer-specific antigen and the T-cell co-receptor CD3. Using antibody single-chain variable region (scFv) sequences derived from amatuximab-recognizing MSLN, and from either blinatumomab or AMG330 targeting CD3, we engineered and expressed two MSLN/CD3-targeting BsAbs: MSLNAMA-CD3L2K and MSLNAMA-CD3AMG, respectively. Both BsAbs promoted T-cell activation and reduced leukemic burden in MV4;11:MSLN xenografted mice, but not in those transplanted with MSLN-negative parental MV4;11 cells. MSLNAMA-CD3AMG induced complete remission in NTPL-146 and DF-5 patient-derived xenograft models. These data validate the in vivo efficacy and specificity of MSLN-targeting BsAbs. Because prior MSLN-directed therapies appeared safe in humans, MSLN-targeting BsAbs could be ideal immunotherapies for MSLN-positive pediatric AML patients.

Role of cytogenetic profiles as prognostic factors for complete remission after induction phase in acute myeloblastic leukemia

Background Risk stratification for acute myeloid leukemia (AML) in children is a must in treatment strategy. This stratification is based on cytogenetic profiles, which are needed to determine proper management to gain better outcomes and reduce side effects of treatment. There is no such risk stratification available in Indonesia until now. Objective To evaluate the association between cytogenetic profiles of t(8,21) and inv(16) mutations with the complete response to induction phase of chemotherapy in pediatric AML. Methods A prospective study was conducted between year 2018 and 2020, involving children with AML from 4 pediatric oncology centers in Jakarta. Subjects were evaluated for cytogenetic profiles, especially t(8,21) and inv(16), as the favorable predictors for AML. Bone marrow remission was evaluated after 2 cycles of induction phase. The results were evaluated for remission rate and survival analysis. Results Karyotype data of 18 subjects were obtained. Translocation t(8;21) detected in 1 subject, and inv(16) mutation in 4 subjects. These two variables had no significant correlation with complete remission after induction phase. Nevertheless, favorable group had more tendencies to achieved remission than unfavorable group. Complete remission achieved in 61% subjects, 90% of theme had a relapse period with an average time 43 weeks. The relapse period in favorable group was shoter than in unfavorable group (34 weeks and 44 weeks, respectively). Conclusions This study shows that cytogenetic profiles of t(8;21) and inv(16) mutation can not be used as prognostic factors for complete remission after induction phase of chemotherapy in pediatric AML.

MLL-SEPT6 Positive Acute Myeloid Leukemia Patients Often Co-occur With NRAS Mutations?

BackgroundThe MLL-SEPT6 fusion gene is a relatively rare genetic event in leukemia. Its clinical characteristics, prognosis, especially the profile of co-occurring gene mutations remain unclear. MethodsWe retrospectively analyzed four rare leukemia cases carrying MLL-SEPT6 in our hospital from laboratory examination, diagnosis, treatment and prognosis, and provided a comprehensive and detailed description on clinical profile of MLL-SEPT6-positive AML patients in the literature. ResultsAll the four patients were diagnosed with acute myeloid leukemia (AML) and harbored X chromosome and 11 chromosome rearrangements. Three of four cases occurred NRAS mutation while the rest one with congenital AML did not. Of the four cases, one developed drug-resistant, one suffered relapse after bone marrow transplantation (BMT) and one died. Combined with other cases reported in literatures, we found that of all patients diagnosed with AML, 90.9% were children (≤ 9 years old) and 54.5% were infants (≤1 year old). The survival time between infant group (≤1 year old) and pediatric group (>1 and <18 years old), patients that received BMT and that received chemotherapy alone did not show significant differences (P>0.05). ConclusionsMLL-SEPT6 was more commonly observed in pediatric AML patients, some of which may co-occur with NRAS mutations. The prognosis was inconclusive and may not be related to age or BMT. More information needs to be accumulated and summarized from additional cases to confirm the underlying connection between NRAS mutations and MLL-SEPT6 in order to better understand the profile in MLL-SEPT6-positive AML.

Evaluating the Efficacy of PRMT5 Inhibitor C220 in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Pediatric acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite maximally intensive therapy, inclusive of chemotherapy and hematopoietic stem cell transplant, approximately 20% of patients experience recurrent disease. These patients are also burdened with treatment-related toxicities. Significant improvements in survival in pediatric AML patients necessitate the incorporation of rational targeted therapies with reduced toxicity. Recent studies demonstrate that PRMT5 knockout or inhibition in syngeneic mouse models of KMT2A (MLL) rearranged leukemic cells increased disease latency (Serio et al., Oncogene, 37:450, 2018; Kaushik et al., Leukemia, 32:499, 2018), indicating that PRMT5 is a potential therapeutic target in pediatric AML. However, there are no reports testing the efficacy of PRMT5 in PDX models of pediatric AML. We evaluated the preclinical efficacy of C220, a potent and selective PRMT5 inhibitor (PRMT5i) (Pastore et al., Cancer Discovery, 10:1742, 2020) in three distinct patient-derived xenograft (PDX) models of KMT2A rearranged AML. Based on the model used for the study, 3-5 million AML cells were injected intravenously in NSG-B2m mice. Disease progression was monitored by evaluating the percentage of human cells in mouse peripheral blood at periodic intervals by flow cytometry. At 2-3 weeks post transplantation, when human cells were detectable in peripheral blood, mice were randomly assigned to control (n=4-5) or treatment (n=2) groups. C220 was administered daily p.o. at a dose of 15 mg/kg for seven days with a break of two days. Mice were dosed with 2-3 additional cycles (indicated in the figure by shaded areas) based on their health status. Mice were monitored daily for experimental endpoints that included body condition score and human cell percentages in peripheral blood. Kaplan-Meier survival plots were generated based on the time when mice were euthanized because they met experimental endpoints. Chronic dosing of C220 prolonged survival and delayed the rise in percentage of human AML cells in mouse peripheral blood in all 3 PDX models (Fig. 1B, D, F). In the NTPL-146 model (KMT2A-MLLT1 fusion), a 135-day improvement in median survival was observed with C220-treatment (Fig. 1A). In the DF-2 (KMT2A-MLLT10 fusion) and DF-5 (KMT2A-MLLT4 fusion) models, which showed a faster engraftment compared to NTPL-146, there was a 5.5-day and 18-day improvement in median survival respectively (Fig. 1C, E). The improvement in median survival was statistically significant in all models (*P&lt;0.05). In conclusion, C220 was effective in controlling leukemia progression and improving survival in KMT2A rearranged PDX models of pediatric AML. Figure 1 Figure 1. Disclosures Gopalakrishnapillai: Geron: Research Funding. Zhang: Prelude Therapeutics: Current Employment. Ruggeri: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barwe: Prelude Therapeutics: Research Funding.

Cite-Seq Reveals Distinct Patterns and Potential Mechanisms of Relapse in Pediatric AML

Introduction - Acute myeloid leukemia (AML) is an aggressive disease with a relapse rate of approximately 40% in children. Progress in improving cure rates has been slow, in part because AML is very heterogeneous. Molecular studies consistently show that most cases are comprised of distinct subclones that diminish or expand over the course of therapy. Single-cell profiling methods now allow parsing of the leukemic population into subsets based on gene and/or protein expression patterns. We hypothesized that comparing the features of the subsets that are dominant at relapse with those that are dominant at diagnosis would reveal mechanisms of treatment failure. Methods - We profiled diagnosis-relapse pairs from 6 pediatric AML patients by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq). All patients were treated at Texas Children's Cancer Center and consented to banking of tissue for research. CITE-Seq was performed by Immunai (New York, NY) using a customized panel of 65 oligonucleotide-tagged antibodies, the 10x Genomics Chromium system for single-cell RNA library generation, and the Novaseq 6000 for sequencing. After data cleanup and normalization, clustering by scRNA-seq was done using the Seurat package. Cell-type identification of clusters was facilitated by published healthy bone marrow scRNA-seq datasets (van Galen et al, Cell 2019). Differentially expressed genes (DEGs) and proteins (DEPs) between diagnosis and relapse were determined using Wilcoxin ranked sum tests. Results - We generated single-cell transcriptomes for a total of 28,486 cells from 12 samples, with a mean of 2373 cells and 1416 genes per sample. Samples were integrated with batch effect correction, producing 30 distinct clusters (cell types) in total (Figure 1A). Cell types with expression profiles consistent with lymphocytes and erythroid precursors were identified in multiple patients, whereas AML cell types tended to be specific to individual patients (Figure 1B). For patients TCH1, TCH2 and TCH3, the most abundant cell types at diagnosis were rare at relapse, and cell types that were rare at diagnosis became dominant at relapse. For these 3 cases, we identified DEGs between the dominant diagnosis cell types and dominant relapse cell types. We found 18 genes that were upregulated at relapse in at least 2 of the cases. Several genes related to actin polymerization were enriched (ARPC1B, ACTB, PFN1), possibly reflecting an enhanced capacity for adhesion and migration. Also of note, macrophage migration inhibitory factor (MIF) and its receptor CD74 were upregulated at relapse, suggesting a role in chemoresistance. For patients TCH4, TCH5 and TCH6, the same cell types that were abundant at diagnosis were also abundant at relapse, and few genes were significantly altered between diagnosis and relapse in multiple cases. Only SRGN, which encodes the proteoglycan serglycin, and GAPDH were altered in 2 of these 3 cases, and both were downregulated at relapse. We performed similar comparisons to identify proteins that were differentially expressed between diagnosis and relapse pairs. The number of DEPs between the dominant diagnosis and relapse cell types ranged from 0 (TCH1 and TCH6) to 5 (TCH2). The only protein altered in more than one case was CD7, which was enriched at relapse in TCH2, TCH3 and TCH4. Conclusions - From CITE-Seq profiling of 6 pediatric AML cases we identified two distinct patterns of relapse. For 3 cases, relapse occurred by expansion of a subset that was small but present at diagnosis. Enrichment of genes associated with adhesion and survival signaling suggests that these cells survived because they were well-equipped to take advantage of interactions with the microenvironment. For 3 other cases, the population that was dominant at diagnosis persisted and expanded at relapse with few substantial changes in gene or protein expression profiles. This pattern suggests that these AML cells were a priori equipped to survive chemotherapy, even though bulk disease levels were transiently reduced below the limit of detection. Most profiled proteins did not change substantially between diagnosis and relapse. An exception is CD7, which was enriched at relapse in 50% of our cases and represents a potential therapeutic target. Analysis of more cases will refine these relapse patterns, reveal potential mechanisms of chemoresistance and inform the development of novel therapies. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P&lt;0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P&lt;0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P&lt;0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P&lt;0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P&lt;0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

Loss of PTEN in Pediatric AML Confers Sensitivity to PARP Inhibition

Pediatric Acute Myeloid Leukemia (AML) is a rare, but deadly cancer. Outcomes over the last 20 years have remained stagnant with an overall 5-year survival rate &lt; 70% and relapse rates around 50%. Further, few new therapies have been successfully introduced to improve these outcomes. Here we report that exploiting deficiencies in DNA damage repair (DDR) is a potential therapeutic strategy for AML. Poly-ADP Ribose Polymerase (PARP) inhibitors were initially developed to target deficient homologous recombination (HR) in BRCA1/2 mutated cancers by blocking single stranded base repair following DNA damage, leading to an accumulation of double stranded DNA breaks, thereby inducing apoptosis. To evaluate the activity of PARP inhibition in pediatric AML, talazoparib was tested as a single agent and in combination with standard chemotherapeutic agents in human AML cell lines representing low (Kasumi-1 and ME-1), intermediate (AML193), and high-risk (CTS, CMS, MOLM-13, and CHRF288-11) disease based on their genomic mutations. Talazoparib showed the highest efficacy as a single agent in all four cell lines with genomic lesions found in high-risk AML subtypes. After combination drug screens, topotecan (synergistic) and gemcitabine (additive) were chosen to move forward to in vivo testing. Our investigational combination was tested in vivo in four murine models representing pediatric AML subtypes harboring AML1-ETO9a (low risk), MLL-AF6 (high risk), CBAF2T3-GLIS2/JAK2 V617F (high risk) and NUP98-KDM5A (high risk) oncogenes. Mice received a backbone of either current standard of care chemotherapy (SOC; anthracycline plus cytarabine) or topotecan plus gemcitabine. NUP98-KDM5A and MLL-AF6 positive mice receiving single agent talazoparib were found to have prolonged survival compared to vehicle alone (p=0.019 and p&lt;0.0001, respectively) which was further enhanced by the addition of chemotherapy irrespective of backbone (p &lt;0.0001). Conversely, mice with AML1-ETOa positive leukemia had no response to single agent PARP inhibitor. While a few mice benefitted from the addition of talazoparib to SOC, this result was not statistically significant (p= 0.42). Early response by bioluminescent imaging confirmed that mice with MLL-AF6 and NUP98-KDM5A driven leukemias who received talazoparib in combination with chemotherapy had the lowest leukemia burdens while the AML1-ETOa cohort did not benefit from the addition of this targeted agent. Interestingly, mice harboring CBAF2T3-GLIS2/JAK2 V617F were not responsive to PARP inhibitors, which was inconsistent with the CMS cell line that has same oncogenic fusion gene but lacks the JAK2 V617F mutation. Synergy experiments with ATM inhibitor AZD0156 demonstrated tremendous synergy with talazoparib in sensitive cell lines with almost no synergy in those that were resistant, suggesting that sensitive cell lines are unable to efficiently activate the HR pathway to repair double stranded breaks induced by PARP inhibition whereas resistant cells can overcome inhibition. To determine the HR response to DNA damage in our cell lines, we exposed them to 1uM topotecan for 2 hours and then measured γH2AX response at 0, 4 and 24 hours. γH2AX is a sensor of DNA damage and therefore increases with DNA damage and decreases with repair. PARP inhibitor sensitive cell lines had persistence of gamma H2AX at 24hrs while resistant cell lines had at least partial resolution of damage, confirming that PARP inhibitor sensitive cell lines have aberrant DNA damage response through HR. RNA sequencing of our cell lines revealed a correlation between Phosphatase and tensin homolog (PTEN) transcript levels and PARP sensitivity. Western blotting confirmed that PTEN was downregulated or absent in both cell lines and murine leukemias that were sensitive to PARP inhibitors. In contrast to the CMS cell line that carries the CBFA2T3-GLIS2 fusion, murine leukemias with CBAF2T3-GLIS2/JAK2 V617F had high levels of PTEN, supporting the hypothesis that sensitivity to PARP inhibitors is due to loss of PTEN. In conclusion, we report that a subset of pediatric AML with high- risk features are sensitive to PARP inhibition due to deficient DDR through HR. Downregulation of PTEN is a candidate biomarker of response to PARP inhibitors in these patients. This data illuminates a promising therapeutic vulnerability in a patient population where new targeted treatments are vital to improve outcomes. Disclosures No relevant conflicts of interest to declare.