New strategies to identify susceptibility to cardiotoxicity by anthracyclines and proteasome inhibitors

Resumen

Background: Long-term cardiotoxicity due to anthracyclines (CDA) is a frequent problem in cancer patients that limits chemotherapy and has repercussions on the prognosis of the oncological disease. Also, chronic CDA has a long-term impact on the quality of life of patients1–3. Anthracyclines produce acute necrosis and apoptosis of cardiomyocytes, leading to myocardial fibrosis and varying degrees of chronic functional damage, even heart failure4,5. The degree of chronic CDA depends on multiple factors that can include dose, age, gender, previous heart diseases, and combined treatment with other drugs such as taxanes, among others6,7. Moreover, since it is difficult to identify which patients will develop chronic CDA, efforts have been made to identify plasma biomarkers. However, current clinical research in Cardio-Oncology has not firmly established biomarkers as useful screening tools for cardiotoxicity in a broad range of populations or cancer therapeutics8,9. By contrast, genetic markers have been used to predict susceptibility to CDA10, as genetics can greatly influence CDA susceptibility. CDA behaves as a complex trait with a polygenic component11. However, the problem of complex traits is how to improve the identification of genetic influence. The proportion of phenotypic variability explained by the genetic component is what is known as heritability. The genetic variants identified associated with complex diseases account only for approximately 10-20 % of the phenotypic variability attributable to genetics. The rest is considered as the missing heritability, and its identification is a matter of ongoing debate12,13. Hypothesis and Objectives: In the first part of the study, we proposed that different levels in the myocardium of molecular pathways (subphenotypes) associated with histopathological heart damage among patients could contribute to different CDA susceptibility. The genes that codified for these protein subphenotypes could help to identify some of the missing heritability14 associated with CDA. Also, these molecular subphenotypes determined in plasma could serve as CDA biomarkers in patients. In the second part of the thesis, we aimed to describe the off-target effect of the proteasome inhibitors alone and in combinations, as in clinics, in hiPSCs-CMs through a contractility and voltage analysis15. In both studies, we aim to identify patients susceptible to develop cardiotoxicity. Patients and Methods: We generated a genetically heterogeneous cohort of 130 mice through a backcross. Mice were treated with doxorubicin or with doxorubicin plus docetaxel once they had developed breast cancer16,17. Histopathological damage after chemotherapy was measured by heart fibrosis and cardiomyocyte size. We quantified in the myocardium levels of genotoxic and signaling pathways, microRNAs, and telomere length, and evaluated their association with histological heart damage. Allelic forms of genes that codify for protein subphenotypes associated with CDA in mice were evaluated in two cohorts of patients treated with anthracyclines. Also, these molecules were quantified in plasma of patients whose degree of CDA was measured by Cardiac Magnetic Resonance18. In the second part of the study, the six proteasome inhibitors tested were: Bortezomib, Carfilzomib, Ixazomib, Oprozomib, Delanzomib and Marizomib and their combinations with two immunomodulators (Lenalidomide and Pomalidomide) and Dexamethasone. The concentrations used were based on the IC50 and Cmax values to mimic human cardiac behavior under treatment conditions. Contractility and voltage analysis were performed with the IC200 instrument (Vala Sciences, California, USA)19. Results: as humans, mice developed greater CDA with aging and with the combined therapy, and there were differences in CDA influenced by genetic background. We identified 16 molecules whose myocardium levels correlated with histopathological damage. The multivariate analysis explained heart damage by the myocardium levels of p70S6K(pT412), γH2AX(S139), P38MAPK(pT180/pY182), CREB(S133) and miRLet7b_5p. In agreement with the polygenic nature of CDA, we identified 80 Quantitative Trait Loci (QTLs) linked to CDA. Among them, as proof of concept, we choose Chr.4-QTL and Chr.11-QTL that were associated with heart damage in different conditions. QTLs linked to protein levels in the myocardium of p70S6K(pT412), γH2AX (S139), and JNK(pT183/pY185) improve the phenotypic variability of heart fibrosis explained by Chr4QTL and Chr11QTL in mice. The genotypes of allelic forms of the RPSKB1 gene that codifies P70S6K, CREB1, and β-TUBULIN genes were associated with CDA. Moreover, plasma quantification of the proteins related to mouse CDA, such as p70S6K(pT412) and JNK(pT183/pY185), was related to functional heart damage and could be used to predict CDA in patients. Regarding the results of the second part of the study, based on the response of the hiPSCs-CMs at Cmax levels, we classified the proteasome inhibitors in three main groups: first, the cardiotoxic proteasome inhibitors that include Bortezomib and Carfilzomib; second, non-cardiotoxic inhibitors: Oprozomib, Ixazomib and Marizomib and third, the most interesting, the combination that seems to have a protective effect against cardiotoxicity at treatment dose: Delanzomib in combination with immunomodulators and dexamethasone. This promising combination is currently in clinical trials. Conclusions: our strategy can identify genetic and plasma markers of CDA and could help to identify patients more susceptible to suffer from this condition; thus, it also could help to choose more individualized chemotherapy. The identification of the genetic component of proteins whose levels in the myocardium are associated with histopathological damage after chemotherapy in a genetically heterogeneous cohort of mice, help to improve the identification of a part of the missing genetic component linked to CDA in mice and humans. Also, the quantification of these molecules in the plasma of patients could serve as CDA biomarkers. The identification of genetic and molecular factors responsible for the increased risk of CDA will eventually contribute to predict and to prevent CDA. Furthermore, the used of hiPSCs-CMs is a promising platform to study and validate the response of the patients to oncological drugs based on the response of a patient’s cells20. In this study, we were able to categorize into three groups: toxic, non-toxic and with a protective effect. Translational implications: Identifying those patients who will develop anthracycline cardiotoxicity is a difficult task. Here we propose a strategy to identify both genetic and plasma biomarkers that help identify those patients. Thus, we treated with anthracyclines a genetically heterogeneous cohort of mice that developed breast cancer. We identified molecules in the myocardium that correlated with histopathological damage. These molecules were considered as molecular subphenotypes of CDA, which is a complex trait. The genes encoding proteins associated with myocardium damage contribute to identifying a part of the genetic component also associated with CDA in patients. Also, those myocardium molecules determined in patients' plasma work as biomarkers to predict CDA and eventually would help to choose more individualized chemotherapy. The use of hiPSCs-CMs allows us to asses the pharmacological off-target effects of new oncological drugs on the heart or predict the tolerance of each patient before starting the treatment. In our study, we were able to classify the drugs into three groups which will help to strengthen the cardiac care in those patients who receive the toxics compounds or increase the dose of non-toxic antineoplastic drugs in refractory patients. Besides, we found that the combination of one of the proteasome inhibitors, currently in a clinical trial with immunomodulators and glucocorticoids, protects against cardiotoxicity. This protective role has a promising path in the future. Testing in hiPSCs-CMs is a new step-through personalized medicine, even though we are still far, nowadays is the best way to recapitulate the effect of the compounds and advance in the cardiotoxicity studies20. References 1. Friedman, M. A., Bozdech, M. J., Billingham, M. E. & Rider, A. K. Doxorubicin cardiotoxicity. 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