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Acute myeloid leukemia (AML) is a heterogeneous disease that accounts for ~20% of all childhood leukemias, and more than 40% of children with AML relapse within three years of diagnosis. Although recent efforts have focused on developing a precise medicine-based approach towards treating AML in adults, there remains a critical gap in therapies designed specifically for children. Here, we present ex vivo drug sensitivity profiles for children with de novo AML using an automated flow cytometry platform. Fresh diagnostic blood or bone marrow aspirate samples were screened for sensitivity in response to 78 dose conditions by measuring the reduction in leukemic blasts relative to the control. In pediatric patients treated with conventional chemotherapy, comprising cytarabine, daunorubicin and etoposide (ADE), ex vivo drug sensitivity results correlated with minimal residual disease (r = 0.63) and one year relapse-free survival (r = 0.70; AUROC = 0.94). In the de novo ADE analysis cohort of 13 patients, AML cells showed greater sensitivity to bortezomib/panobinostat compared with ADE, and comparable sensitivity between venetoclax/azacitidine and ADE ex vivo. Two patients showed a differential response between ADE and bortezomib/panobinostat, thus supporting the incorporation of ex vivo drug sensitivity testing in clinical trials to further evaluate the predictive utility of this platform in children with AML.

Children with Down syndrome have an approximately 10-fold increased risk of developing acute lymphoblastic leukemia and this risk is influenced by inherited genetic variation. Genome-wide association studies have identified IKZF1 as a strong acute lymphoblastic leukemia susceptibility locus in children both with and without Down syndrome, with association signals reported at rs4132601 in non-Down syndrome and rs58923657 in individuals with Down syndrome (r2 = 0.98 for these two loci). Expression quantitative trait locus analysis in non-Down syndrome lymphoblastoid cell lines has demonstrated an association between the rs4132601 risk allele and decreased IKZF1 mRNA levels. In this study, we provide further mechanistic evidence linking the region encompassing IKZF1-associated polymorphisms to pro-leukemogenic effects in both human lymphoblastoid cell lines and murine hematopoietic stem cells. CRISPR/Cas9-mediated deletion of the region encompassing the rs17133807 major allele (r2 with rs58923657 = 0.97) resulted in significant reduction of IKZF1 mRNA levels in lymphoblastoid cell lines, with a greater effect in Down syndrome versus non-Down syndrome cells. Since rs17133807 is highly conserved in mammals, we also evaluated the orthologous murine locus at rs263378223, in hematopoietic stem cells from the Dp16(1)Yey mouse model of Down syndrome as well as non-Down syndrome control mice. Homozygous deletion of the region encompassing rs263378223 resulted in significantly reduced Ikzf1 mRNA, confirming that this polymorphism maps to a strong murine Ikzf1 enhancer, and resulted in increased B-lymphoid colony growth and decreased B-lineage differentiation. Our results suggest that both the region encompassing rs17133807 and its conserved orthologous mouse locus have functional effects that may mediate increased leukemia susceptibility in both the Down syndrome and non-Down syndrome genetic backgrounds.

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CAS Article Google Scholar Download references This work was supported by an American Society of Hematology Scholar Award (JJJ); grant RP170074 from the Cancer Prevention and Research Institute of Texas (KRR); funding from the Lynch family (KRR); and by the National Cancer Institute, National Institutes of Health (R01 CA207086 to HDL). Flow cytometry assays were performed at the Research Flow Cytometry Core Facility of Texas Children’s Cancer and Hematology Centers with the support from the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574), and the assistance of Joel M. Sederstrom. Flow assistance was also generously provided by Amos Gaikwad and Tatiana Goltsova. Histology was performed by the BCM Center for Comparative Medicine Comparative Pathology Laboratory with the assistance of Brian Simons. Microscope assistance was generously provided by Debananda Pati and Nenggang Zhang. Texas Children’s Cancer Center, Department of Pediatrics, Baylor College of Medicine (BCM), Houston, TX, USA Jacob J. Junco, Raushan Rashid, Maci Terrell, Vincent U. Gant Jr., Matthew Miller, Rachel Rau & Karen R. Rabin Department of Pathology & Immunology, BCM, Houston, TX, USA Taylor Chen & H. Daniel Lacorazza Stem Cells and Regenerative Medicine Center, Departments of Pediatrics and of Molecular & Human Genetics, BCM, Houston, TX, USA Matthew Miller & Rachel Rau You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar You can also search for this author in PubMed Google Scholar Correspondence to Jacob J. Junco. The authors declare that they have no conflict of interest. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions Junco, J.J., Chen, T., Rashid, R. et al. An Mb1-Cre-driven...

Background Relapse free survival of pediatric AML remains only 60%. Current standard myelosuppressive therapy has been maximized, so novel therapies with minimal toxicities are needed to improve outcomes. Previously, we found atovaquone (AQ), an FDA-approved drug that treats pneumocystis jiroveci pneumonia (PJP), reduces AML burden - by suppressing oxidative phosphorylation (OXPHOS) - in xenograft mice. Clinically achievable concentrations of AQ for anti-PJP are 40-80µM, but the anti-leukemia effects are observed as low as 10µM (Stevens et al, Bld Adv, 2019). This makes AQ an ideal drug to incorporate into AML treatment. AQ is a daily administered oral medication, and plasma levels depend on patient compliance, absorption, and entero-hepatic recirculation, which can be compromised due to the patient population and adverse events (AE) of chemotherapy. Here we investigated the feasibility of incorporating AQ into standard pediatric AML treatment. Methods Patients with de novo AML were enrolled at two children's hospitals in the USA. Daily administration of AQ at established PJP prophylaxis dosing was combined with standard chemotherapy for AML, based on the Medical Research Council (MRC) backbone of cytarabine 100mg/m2 q12h x 10 days, and daunorubicin 50mg/m2/dose on days 1, 3, and 5. As it was unclear if our AQ dosing would provide adequate PJP prophylaxis, it was left to provider discretion to give additional PJP protection. AQ compliance, AEs (per NCI CTCAE v5), parent/caregiver ease of administration score (scale: 1-5, 1=very difficult, 5=very easy to administer) and peripheral blood/bone marrow pharmacokinetics (PK) were collected during Induction 1. Real time AQ plasma concentration results were not provided. To address feasibility of achieving adequate levels, all gastrointestinal (GI) AEs ≥ grade 2 were collected, in addition to grade 4 or greater AEs. Patients who took at least 85% of planned doses and missed less than 2 consecutive doses of AQ were eligible for analyses. Correlative biology studies assessed AQ induced apoptosis at 30uM, effects on OXPHOS and relevant signaling activities. Patient derived xenografts (PDX) were established and treated with AQ. This trial is registered with ClinicalTrials.gov (NCT03568994). Results A total of 26 pediatric AML patients enrolled (ages 8 months - 19.7 years, mean 10.7 years); 24 patients were evaluable for this study. Two patients had Grade ≥ 3 GI toxicities that prohibited enteral administration so they were excluded from AQ PK and ease of administration analyses. We found that 14/24 (58%) patients achieved plasma levels above the target anti-leukemia concentration (10µM) by day 11. At the end of induction, 19/24 (75%) patients achieved plasma levels above 10µM, but only 7/24 (29%) patients achieved adequate levels for PJP prophylaxis (40µM). Only 1 patient achieved levels above 40µM throughout the trial [FIG A]. Mean ease of administration score was 3.8. For the youngest patients (x ≤ 2.6years), the average score was 3.4 which was not significantly different from older patients (ANOVA, p > 0.05) [FIG B]. Ease of administration scores showed no association with plasma levels (Pearson's correlation, p > 0.05). Finally, correlative biology studies in patient samples demonstrated robust AQ-induced apoptosis, OXPHOS suppression, and prolonged survival in a PDX model receiving AQ [FIG C]. Conclusion Our data demonstrate the feasibility of combining AQ with traditional chemotherapy for pediatric AML. Patients of all ages were able to tolerate AQ and no AEs were attributable to AQ administration. The target anti-leukemic concentration of AQ in the plasma (> 10uM) was frequently achieved, but concentrations of > 40uM at standard dosing were rare. Low plasma levels of AQ did not correlate with the presence of GI related AEs or weight loss, so plasma levels should be monitored to ensure sufficient PJP prophylaxis. Our correlative biology results support suppression of OXPHOS as the primary mechanism of action by which AQ exerts its anti-leukemia effect, and AQ may be an active anti-leukemia agent for pediatric AML patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Pediatric acute myeloid leukemia (AML) is a rare disease with roughly 600 cases diagnosed in the United States each year with minimal improvement in clinical outcomes over the last few decades. We previously demonstrated that an ex vivo drug sensitivity assay (DSA) can predict clinical response in myelodysplastic syndrome (Spinner et al. Blood Adv 2020). Here we investigated whether the DSA performed on pre-induction pediatric AML samples correlates with clinical response and can identify potent novel drug combinations. Methods Pre-induction blood or bone marrow samples were assayed from 20 de novo pediatric AML patients diagnosed at Texas Children's between 5/2015 and 10/2020. All patients consented to research (82% enrolled in clinical trial identifier NCT03568994) and received ADE (Cytarabine, Daunorubicin, and Etoposide), and next-generation sequencing was done as part of clinical care. Risk stratification was per AAML1831 guidelines. Drug sensitivity data was analyzed from 13/20 samples that passed quality control with matched treatment conditions: 9/13 (69%) patients had M1/M2 histology, 3/13 (23%) were M4/M5 and 1/13 (8%) was M7 with a median age of 12.3 years. For the ex vivo DSA, samples were incubated in conditioned media and treated with a single dose of up to 25 unique compounds and up to 149 drug combinations. After 72 hours, changes in tumor blast populations were assessed by flow cytometry using an 11-marker panel to identify blasts. For each treatment condition, drug sensitivity was calculated based on the number of blasts remaining after treatment compared to DMSO control. Clinical response data, including minimal residual disease (MRD) percentage by flow cytometry, and 1-year relapse-free survival (RFS), were correlated with drug sensitivity results. Log odds ratios (OR) were calculated with the Haldane-Anscombe correction. ORs were used to quantitatively measure the association between clinical attributes and the DSA to the clinical response data. For evaluation of ORs, a normalized blast score of 70% viability was used to maximize the separation between high and low drug sensitivity. Results Ex vivo drug sensitivity correlated with both MRD (r=0.63) and 1-year RFS (r=0.59) in the de novo patient subset (Fig A). Three patients with an MRD >1% exhibited low ex vivo sensitivity to ADE, and among these 3 patients, 2 did not achieve 1-year RFS. Results from the DSA predicted increased odds of having an MRD >1% compared to demographic and mutational clinical attributes that showed weaker associations with MRD (Fig B). Of the 77 treatment conditions that were tested in 13 patient samples, Bortezomib in combination with Panobinostat (B/P) was the most efficacious treatment in the DSA, where drug sensitivity ranged from low (>100% blast viability) to high (0% blast viability). Separation of patient samples into two distinct low and high DSA response groups was observed with B/P, whereas ADE and single agents showed a graded distribution (Fig C). Within these response groups, pAML3 showed low sensitivity to ADE in the ex vivo DSA and the patient did not respond to ADE. In contrast, pAML8 showed high sensitivity to ADE ex vivo and the patient responded to ADE treatment. While pAML3 and pAML8 showed similar ex vivo sensitivity to B/P as for ADE (Fig D), pAML4 showed preferential sensitivity to ADE and not B/P, and conversely pAML6 showed sensitivity to B/P and not ADE. Conclusion Ex vivo drug sensitivity to ADE correlates with both MRD and 1-year RFS in a cohort of 13 de novo pediatric AML patients. These results suggest that clinical response in pediatric AML may be assessed prior to treatment using an ex vivo drug sensitivity assay. Compared to demographic and mutational clinical characteristics queried, ex vivo drug sensitivity to ADE has the potential to be a more predictive measure compared to clinical attributes alone. Combining genomics with functional ex vivo drug sensitivity data could further enhance precision medicine and biomarker discovery in pediatric AML. The DSA also highlights Bortezomib/Panobinostat as a potential novel drug combination for pediatric AML, and the ability to identify a patient sample that is insensitive to ADE and sensitive to Bortezomib/Panobinostat ex vivo supports the use of the DSA to not only predict clinical response but also to possibly inform treatment decisions for pediatric AML patients. Figure 1 Figure 1. Disclosures Strachan: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Gu: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Richardson: Notable Labs: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Anderson: Notable Labs: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Patents & Royalties.

10032 Background: Pediatric acute myeloid leukemia (AML) is a rare disease with roughly 500 cases diagnosed in the United States each year and has had minimal improvement in clinical outcomes over recent decades. Novel treatment development to improve outcomes may be enhanced with an accompanying test for predicting treatment response. We previously demonstrated that an ex vivo drug sensitivity assay (DSA) can predict clinical response in myelodysplastic syndrome. Here we investigated the use of the DSA in pediatric AML patients, including a subset participating in a clinical trial of atovaquone. Atovaquone is an FDA-approved anti-parasitic drug that was associated with lower relapse rates in adult AML patients. Adding atovaquone and other standard of care combination treatments into the DSA, we investigated whether the assay, performed on pre-induction samples, correlated with measures of clinical response. Methods: We assayed pre-induction blood or bone marrow samples from 22 de novo pediatric AML patients diagnosed at Texas Children’s between 5/2015 and 10/2020 who consented to research (82% enrolled in clinical trial identifier NCT03568994). We subsetted this analysis to patients who received ADE (Cytarabine, Daunorubicin, Etoposide) (n = 20) induction, with the majority additionally receiving atovaquone (n = 16). For the DSA, samples were incubated with up to 25 compounds, including the treatment drug combinations and each of the compounds individually. After incubation, changes in tumor blast populations were assessed by flow cytometry. For each drug condition, drug sensitivity was calculated based on the number of blasts remaining after treatment. After quality control, downstream analyses were limited to 13 samples. Clinical response data, including minimal residual disease (MRD) percentage by flow cytometry and one year relapse-free survival, were correlated with the drug sensitivity results. Results: For the de novo subset analysis, we observed correlations between ex vivo drug sensitivity with both MRD percentage (r = 0.63) and one year relapse-free survival (RFS1, AUC = 0.92). The 3 patients with lowest ex vivo sensitivity had the highest MRD percentages (mean 21%). 2 of the 3 patients who did not achieve one year relapse-free survival had the lowest ex vivo sensitivity. Drug combination sensitivity correlated more with MRD and RFS1 than the single agents alone (single agent mean MRD r = 0.39). Conclusions: In our cohort, ex vivo DSA for ADE and atovaquone in pediatric AML cases correlated with both MRD and one year relapse-free survival. This suggests that clinical response in pediatric AML may be assessed prior to treatment using a DSA. This study also suggests that the DSA can be used to test drug combinations, and thus may be used for investigating novel treatment combinations. Further development of the DSA may benefit treatment decisions and prioritization of drug development.