Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

Keywords: Cancer immunotherapy; Drug therapy; Leukemias; Oncology; Therapeutics.

Figure 1. HOSU-53 is a selective DHODHi.

(A) Chemical structure of HOSU-53. (B) In vitro uridine rescue assay using MOLM-13 AML cell line. Combenefit software was used to evaluate highest single agent synergy and antagonism between increasing concentrations of HOSU-53 and exogenous uridine (n = 3). (C) In vivo pharmacokinetics (PK) and pharmacodynamics (PD) modeling after single-dose administration of intravenous (i.v.) or oral (p.o.) HOSU-53 in wild-type male mice (n = 3/group). Data shown as mean and error SD.

Figure 2. HOSU-53 demonstrates in vitro potency and differentiation properties in AML.

(A) MTS proliferation assay using primary AML samples (n = 8) to determine the 50% inhibitory concentration (IC₅₀) of HOSU-53 after 96-hour treatment. Data shown as mean and error SD. GraphPad Prism was used to analyze, visualize, and calculate IC₅₀ values. (B) Representative hema 3 differential staining (Thermo Fisher Scientific) of cytospin preparation slides for a primary AML sample treated with HOSU-53 in vitro for 7 days (long-term culture, LTC) to determine morphology changes. Images were taken using BioTek Cytation 5 Cell Imaging Multimode Reader at 20× original magnification. (C) Representative flow cytometry histogram plot for in vitro E. coli phagocytosis assay using THP-1 cells treated with HOSU-53 for 6 days. Cytochalasin D (Cyto. D), an actin polymerization inhibitor was used as an inhibitor of phagocytosis. Phagocytosis assay experiments were done 3 independent times.

Figure 3. The use of DHO plasma concentration to gauge HOSU-53 tolerability and toxicity.

(A) Multiple ascending dose study to determine the maximum in vivo daily tolerated oral dose of HOSU-53. Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive increasing HOSU-53 daily p.o. doses. ^aMice in the 30 mg/kg group showed weight loss by day 13 of treatment. Thus, a dosing holiday was instituted followed by resuming treatment on day 18 at 20 mg/kg reduced dosage. ^bMice in the 100 mg/kg groups showed weight loss instituting a dosing halt on day 6 of treatment. Acceptable tolerability was defined as a group mean body weight (BW) loss of less than 20% during the study. Any dosing regimen resulting in greater BW loss was considered above the maximum tolerated dose (MTD). Adjusted FDR P value *<0.05, ***≤0.0001. (B and C) Correlative efficacy and PK/PD in vivo analysis of HOSU-53. Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group for survival study, n = 3/group for PK/PD analysis) were enrolled to receive increasing HOSU-53 oral doses to correlate efficacy (B) with the plasma concentration of HOSU-53 (PK) and on-target metabolite, DHO accumulation (PD) at day 1 and cumulative day 14 (C). Green dotted line represents the hypothesized threshold of tolerability. Data represent individual values of each mouse. Adjusted FDR P value ***≤0.0001. qd, daily; biwk, twice weekly.

Figure 4. HOSU-53 results in superior in vivo outcome using AML disseminated xenograft model.

(A) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive daily p.o. 4 mg/kg of HOSU-53 or BAY to compare with (B) daily p.o. 10 mg/kg of HOSU-53 or BAY and determine using dose-matched regimens the overall survival after HOSU-53 versus BAY, a potent clinical DHODHi candidate. In the 4 mg/kg and 10 mg/kg BAY groups, 1/10 mice were excluded from analysis because of non-treatment-related accidents: gavage error and animal not found in cage, respectively. Figure 3B and A and B share the same vehicle and HOSU-53 arms. Adjusted FDR P value **≤0.001, ***≤0.0001.

Figure 5. HOSU-53 significantly enhances the outcome of select FDA-approved AML therapies.

(A) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 10 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive daily p.o. 4 mg/kg or 10 mg/kg HOSU-53 to compare their efficacy with daily p.o. 30 mg/kg gilteritinib (gilt) FLT3 inhibitor or the combination of 4 mg/kg HOSU-53 with gilt. In the gilt combination cohort, 4 mice were euthanized for tissue harvest to determine disease burden, and the remaining 6 mice were kept on study for survival analysis. Black arrow indicates treatment was stopped at day 79. Adjusted FDR P value ***≤0.0001. Vehicle and HOSU-53 arms in this study are shared with Figure 8, A and B. (B) Using the P53-null HL-60 CDX tumor–bearing model, 14 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive 0.4 mg/kg decitabine (Dec) hypomethylating agent (HMA) i.p. as 4 days on/10 days off cycles or daily p.o. 10 mg/kg HOSU-53 or combination of both agents. Dotted black line indicates treatment was stopped and end of study at day 82. P value *≤0.05, ***≤0.0001. (C) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive daily p.o. 4 mg/kg HOSU-53 to compare its monotherapy efficacy with 1.5 mg/kg azacitidine (aza) HMA i.p. for 5 days every 16 day cycles monotherapy or in combination with daily p.o. 25 mg/kg venetoclax (aza/ven) or daily p.o. 4 mg/kg HOSU-53 (aza/HOSU-53). Adjusted FDR P value ***≤0.0001. Supplemental Figure 4 shows all 9 groups done in this study; herein we show the most significant regimens for clarity.

Figure 6. HOSU-53 significantly prolongs survival in a PDX in vivo tumor model.

(A) Using a relapsed or refractory (r/r) MLL/AF10 rearranged, therapy-related AML passaged PDX sample (CCHMC-2017-14), NRGS mice (n = 9–10/group) were intravenously engrafted with 5 × 10⁵ cells/mouse. At 13 days after engraftment, mice were enrolled to receive vehicle, 10 mg/kg HOSU-53 orally 5 days each week, 0.5 mg/kg azacitidine (aza) i.p. 4 days each week for 4 weeks, or combination of aza and HOSU-53. Mice were continually monitored till end removal criteria. (B) On day 27 after engraftment (D27), a bone marrow aspirate (BMA) was performed to assess disease burden by measuring the surface expression of human CD45 on live cells (7-AAD–negative cells) using flow cytometry. Data shown as scatter dot plot mean with SD. Adjusted FDR P value *<0.05, ***≤0.0001. (C) Representative Wright-Giemsa differential staining (Thermo Fisher Scientific) for the D27 BMA cells cytospin preparation slides. Images were taken using BioTek Cytation 5 Cell Imaging Multimode Reader at 40× original magnification.

Figure 7. DHODHi mediate the modulation of CD38 surface expression, resulting in synergy with anti-CD38 therapies.

(A) Representative flow cytometry overlay histogram plot for CD38 surface expression in MOLM-13 cell line following a 72-hour treatment with BRQ or increasing concentrations of HOSU-53 in the presence or absence of 0.1 mM uridine (U) supplementation (experiment was done 3 independent times). (B and C) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 8/group) were enrolled to receive daily p.o. 10 mg/kg HOSU-53 or biwk 1 mg/kg i.p. daratumumab (Dara) (B) or biwk 2.5 mg/kg i.p. isatuximab (Isa) (C) anti-CD38 antibodies or a combination of HOSU-53 and Dara (B) or combination of HOSU-53 and Isa (C). B and C share same vehicle and HOSU-53 monotherapy arms, with data split into 2 figures for clarity. Adjusted FDR P value *<0.05.

Figure 8. HOSU-53 in combination with anti-CD47 therapy results in long-term disease-free survival in the MOLM-13 CDX AML in vivo model.

(A) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive vehicle, daily p.o. 4 mg/kg of HOSU-53, i.p. 0.5 mg/animal daily for 21 days of CD47 antibody (clone B6H12, BioXCell), or combination of both. Arrows indicate stopping treatment on day 80 and end of study on day 106. In the combination arm, 5 of 10 mice were euthanized for bone marrow collection. On end of study, day 106, the surviving 4 mice were euthanized for bone marrow collection. Adjusted FDR P value ***≤0.0001. (B) Using the FLT3-mutant MOLM-13 CDX tumor–bearing model, 4 days after i.v. engraftment, NCG mice (n = 10/group) were enrolled to receive vehicle, daily p.o. 10 mg/kg of HOSU-53, i.p. 0.5 mg/animal daily for 21 days of CD47 antibody (clone B6H12, BioXCell), or combination of both. Arrows indicate stopping treatment on day 80 and end of study on day 106. In the combination arm, 4 of 10 mice were euthanized for bone marrow collection. On end of study, day 106, the surviving 4 mice were euthanized for bone marrow collection. Adjusted FDR P value *<0.05, ***≤0.0001. A and B share the vehicle and CD47 antibody arms. (C) Representative flow cytometry plot demonstrating the lack of residual human CD45 in bone marrow samples harvested at day 80, end of treatment. (D) Representative flow cytometry plot demonstrating the lack of residual human CD45 in bone marrow samples harvested at day 106, end of study.