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Recurrence and drug resistance are major challenges in the treatment of acute myeloid leukemia (AML) that spur efforts to identify new clinical targets and active agents. STAT3 has emerged as a potential target in resistant AML, but inhibiting STAT3 function has proven challenging. This paper describes synthetic studies and biological assays for a naphthalene sulfonamide inhibitor class of molecules that inhibit G-CSF-induced STAT3 phosphorylation in cellulo and induce apoptosis in AML cells. We describe two different approaches to inhibitor design: First, variation of substituents on the naphthalene sulfonamide core allows improvements in anti-STAT activity and creates a more thorough understanding of anti-STAT SAR. Second, a novel approach involving hybrid sulfonamide–rhodium(II) conjugates tests our ability to use cooperative organic–inorganic binding for drug development, and to use SAR studies to inform metal conjugate design. Both approaches have produced compounds with improved binding potency. In vivo and in cellulo experiments further demonstrate that these approaches can also lead to improved activity in living cells, and that compound 3aa slows disease progression in a xenograft model of AML.

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.

Atovaquone, a US Food and Drug Administration–approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 mM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 ,30 mM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2a, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2a led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.