Annamycin, a Non-Cardiotoxic Anthracycline Demonstrates Unique Organotropism and Activity Against Ara-C and Venetoclax Resistant AML
Authors: Zielinski Rafal Jerzy , Grela Krzysztof , Cardenas-Zuniga Roberto , Skora Stanislaw , Fokt Izabela , Gagea Mihai , Andreeff Michael , Baran Natalia , Priebe Waldemar 
Published: December 2024

Background:

Anthracycline-based therapies constitute the backbone of intensive chemotherapy regimens for patients with acute myeloid leukemia (AML) due to their effectiveness in reducing tumor burden. However, cardiotoxicity and drug resistance limit their clinical use. This hurdle inspired us to develop novel highly efficacious agents with improved safety profiles, such as Annamycin (ANN), a doxorubicin (DOX) analog. ANN is undergoing clinical evaluation as a liposome formulated drug (L-ANN) in AML and sarcoma patients. Our previous preclinical studies demonstrated ANN's anti-leukemic activity, which is not cross-resistant with that of DOX, and lack of cardiotoxicity in vivo. We aimed to directly assess the impact of L-ANN and its unique organotropism on eradication of leukemia as a single agent and in combination with cytarabine (Ara-C) and Venetoclax (VEN) in sensitive and resistant cell lines. Next, we compared the impact of ANN and DOX on physiology of rat and human cardiomyocytes. Further, we assessed in vivo cardiotoxicity in mice following chronic exposure to L-ANN or DOX at therapeutic and MTD doses.

Methods:

Subsets of parental, Ara-C-resistant, and VEN-resistant AML cell lines were treated with ANN at 0-3000 nM in vitro, alone, or ± VEN (1-1000 nM) and ± Ara-C (1-3000 nM). Treatment of naïve and heavily pretreated relapse/refractory primary AML patient samples were also evaluated. The impact of DOX and L-ANN was further tested on established cultures of rat H9c2 cardiomyoblasts derived from ventricular tissue of myocardium and on human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). RTCA CardioECR was applied to probe-free determination of viability (cell index, impedance), contractility, and electric potential. Finally, anti-leukemic efficacy of L-ANN in combination with Ara-C was evaluated in an aggressive, TP53 null FLT3-ITD mutated syngeneic AML Turqoise2 model, with extensive evaluation of tumor burden in bone marrow, spleen, lungs, and liver by fluorescence imaging. PK and tissue-organ distribution of ANN were analyzed in naïve mice and rats versus DOX.

Results:

L-ANN displayed synergy with Ara-C and VEN in reducing viability in parental treatment naïve cell lines (10-20 nM) and in Ara-C-and VEN-resistant cell lines (30-350 nM). L-ANN showed no apparent toxicity in vivo. Parallel comparison of L-ANN and DOX at 8 mg/kg for 7 weeks exhibited a favorable toxicity profile for L-ANN, with no evidence of cardiotoxicity ex vivo. DOX treated mice demonstrated significant weight loss and increased levels of lactate dehydrogenase (LDL) in blood serum. Histopathological evaluation of heart tissue postmortem revealed mild cytoplasmic vacuolation of cardiac myocytes only in DOX-treated cohorts. Evaluation of human cardiomyocytes treated with ANN or DOX revealed a limited impact of ANN on human cardiomyocyte contractility, viability, and electric potential up to the highest tested dose of 1.5 uM as assessed by RTCA, in opposition to heavily perturbed contractility induced by DOX at 0.5 uM. Finally, L-ANN's ability to extend survival was potentiated in combination with Ara-C. L-ANN was well tolerated by the animals even at schedules exceeding the therapeutic dosage of 4 mg/kg. Ex vivo pathology examination confirmed no toxicity to the murine heart/myocardium, similar to patients in clinical trials.

PK and tissue-organ distribution of L-ANN revealed significantly higher concentrations of ANN vs. DOX in leukemia homing organs, suggesting conditions that might contribute to increased therapeutic efficacy and reduced MRD.

Finally, assessment of L-ANN administration resulted in durable disease eradication up to 150 days post treatment in 20% of mice. Interestingly, rechallenging these animals with AML-Turq-2 cells resulted in extended survival compared to naïve mice, suggesting immune-memory inducing properties of L-ANN therapy and warranting further examination.

Conclusion:

This study confirms that L-ANN effectively targets both Ara-C and VEN resistant AML cell lines and heavily pretreated Relapse/Refractory primary AML patients in vitro. Furthermore, lack of apparent cardiotoxicity, improved organotropism, synergy with Ara-C, and possible immune-memory reinforcing properties contributes to the favorable performance of L-ANN in clinical settings (NCT03388749; NCT05319587).