Tumour initiated purinergic signalling promotes cardiomyocyte RBFOX1 degradation and cardiotoxicity from DNA damaging anticancer agents

Figures

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  Fig. 1. Inosine and hypoxanthine can predict cardiotoxicity susceptibility in cancer patients and its levels can be regulated by the stemness transcription factor ZNF281 which can stabilize several purinergic factors on the cell membrane of cancer cells. a Under normal conditions, (1) terminally differentiated cardiomyocytes have condensed inaccessible chromatin that we hypothesise becomes (2) more accessible by tumour secreted factors, promoting loss of cardiomyocyte maturity and (3) intercalation of DNA damaging chemotherapeutics into now accessible chromatin resulting in DNA breaks and cardiotoxicity (BioRender Lorenzana (2025) https:// BioRender.com/2dyaqis). b Graph shows baseline cumulative inosine and hypoxanthine plasma levels in breast cancer patients from the MANTICORE trial placebo arm. Patients are grouped above or below a threshold of 6 μM: no cardiotoxicity (teal), mild cardiotoxicity (lilac) and moderate cardiotoxicity (purple), n = 22 patients, *P = 0.036 comparing CIC and no CIC above and below a post-hoc analysis determined threshold of 6 μM in a 2 × 2 contingency table, P-value is based on Fisher’s exact two-sided test. c Extracellular purinergic signalling and…

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  Fig. 2. Inosine and hypoxanthine can bind and activate the A2A receptor on cardiomyocytes to promote a CAMKIIδ-mediated RBFOX1 phosphorylation and caspase-1-mediated degradation. a Immunoblots show T286P-CAMKIIδ and RBFOX1 after A2A agonists treatment, n = 3 biological replicates, *P = 0.05 compared to vehicle. b Immunoblots show GFP, T286P-CAMKIIδ and RBFOX1 after adenovirus treatment overexpressing GFP or CAMKIIδ, n = 3 biological replicates, *P = 0.05 compared to GFP. c Immunoblots show T286P-CAMKIIδ and RBFOX1 after A2A agonist UK-432097 dose-response, n = 3 biological replicates, *P = 0.05 compared to vehicle. d Immunoblots show T286P- CAMKIIδ and RBFOX1 after CAMKII inhibitor KN93 ± inosine, *P = 0.0143 compared to vehicle, #P = 0.0143 compared to inosine. e Immunoblots show RBFOX1 in A549 cells transfected with empty vector, RBFOX1 WT, T197E RBFOX1 ± caspase 1 inhibitor (YVAD-CMK) or pan-caspase inhibitor (YVAD-CHO), n = 3 biological replicates, *P = 0.05 compared to WT, #P = 0.05 compared to T197E. f Immunoblots show RBFOX1 after UK-432097 ± YVAD-CMK treatment, *P = 0.0286 compared to vehicle, #P = 0.0143 compared to UK-432097. g Boyden chamber with A549 cancer cells plated above…

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  Fig. 3. Loss of RBFOX1 in cardiomyocytes can promote a less mature state and GRB7 loss mediated activation of the mitochondrial permeability transition pore. a Immunoblots show RBFOX1, MEIS1, HOXB13, RUNX1, DAB2, K9Ac-H3, S10P-H3, and S33P- RPA in control Cre+ and RBFOX1 KD Cre+, n = 5 mice, all comparisons **P = 0.004 except MEIS1, **P = 0.0079 compared to control Cre+ where RBFOX1 and MEIS1, HOXB13, RUNX1, DAB2 and K9Ac-H3, S10P-H3, S33P- RPA are ran on separate gels and processed in parallel. b Immunoblots show RBFOX1, MEIS1, HOXB13, DAB2, and K9Ac-H3 in adult compared to foetal mouse heart tissue, n = 2 mice where RBFOX1 and MEIS1, HOXB13, DAB2 and K9Ac-H3 are ran on separate gels and processed in parallel. c mPTP assay measuring calcein AM fluorescence (green) in adult control Cre+ and RBFOX1 KD Cre+ cardiomyocytes ± cyclosporin A for 30 minutes (CSA, a mPTP inhibitor), n = 3 biological replicates, *P = 0.05 compared to control Cre+, #P = 0.05 compared to RBFOX1 KD Cre+. d Immunoblots show GRB7 in cardiomyocytes isolated from control Cre+ and RBFOX1 KD Cre+ mice, n = 5 mice, **P = 0.004 compared to control Cre+. e Immunoblots show RSVGRB7, TUBULIN, MFN2, LAMIN A/C in cellular…

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  Fig. 4. Klkb1-ZNF281 overexpression transgenic or cardiomyocyte-specific RBFOX1- deficient mice develop more severe cardiotoxicity and dilated cardiomyopathy, compared to controls. a Immunoblots show RBFOX1, DAB2, HOXB13, RSVGRB7, K9Ac-H3, S10P-H3, and S33P-RPA from Control and ZNF281 OE, n = 5 mice, *P = 0.0159 for DAB2, *P = 0.0278 for S33P-RPA and **P = 0.004 for all others compared to Control where each target is run on a separate gel and processed in parallel except RBFOX1, HOXB13 and S10P-H3, S33P-RPA. b Comet assay in control vs. ZNF281 OE or (c) control Cre+ vs. RBFOX1 KD Cre+ cardiomyocytes treated with doxorubicin (1 μM) for 72 h (mean data in Supplementary Fig. 14). d Immunoblots show P53, APAF1, cleaved caspase 9 (c-CAS 9) from control and ZNF281 OE or (e) Control Cre+ and RBFOX1 KD Cre+ mice after chemotherapies: doxorubicin (1 μM), epirubicin (1 μM), cisplatin (500 nM), etoposide (500 nM), cyclophosphamide (500 nM), n = 3 biological replicates, *P = 0.05 compared to paired Control or control Cre+ chemotherapy. f Control and ZNF281 OE or (g) Control Cre+ and RBFOX1 KD Cre+ mice received doxorubicin and cardiac function was assessed via echocardiography using ejection…

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  Fig. 5. Loss of RBFOX1 and activation of the apoptosome are features in explanted myocardial tissues of anthracycline-induced cardiotoxicity patients. a Immunoblots show RBFOX1, P53, APAF1, c-CAS9 in a well characterised cohort of non- failing and AIC human patient explanted heart tissues. b Quantification values are expressed as mean ± SEM, n = 10 and 12 different patients. ***P = 0.0006 for RBFOX1, ***P = 0.0002 for c-CAS9,****P < 0.0001 for P53 and APAF1 compared to non-failing patients, P values are based on one-sided Mann-Whitney Test. Individual ACTIN was used as a loading control for each target run on different gels and processed in parallel. c Spearman’s rank order correlation shows RBFOX1 correlates to P53 negatively r = -0.8306, P < 0.0001, to APAF1 negatively r = -0.7628, P < 0.0001, to c-CAS9 negatively r = -0.6623, P = 0.0004. Source Data file is provided.

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  Fig. 6. Mechanism for inosine and hypoxanthine mediated cardiomyocyte RBFOX1 degradation and loss of maturity. Tumour ZNF281 mediates purinergic signalling and secreted inosine and hypoxanthine can bind and activate the cardiomyocyte A2A receptor, which results in phosphorylation and activation of CAMKIIδ, which can phosphorylate and promote caspase-1 mediated degradation of RBFOX1 (HIT 1). RBFOX1 loss results in decreased levels of RSVGRB7 and increased mPTP assembly and activity and release of cytosolic cytochrome c, as well as epigenetic remodelling via phosphorylated and acetylated H3, resulting in an open chromatin state. At this stage, several DNA damaging agents can intercalate with cardiomyocyte DNA, causing DNA breaks and induction of the pro-apoptotic transcription factor P53, along with its downstream transcriptional target APAF1 (HIT 2). Cytosolic cytochrome c can now bind APAF1 to initiate apoptosis and cardiomyocyte cell death, resulting in cardiotoxicity.