Investigating the anti-leukaemic effect of tamoxifen in the myeloproliferative neoplasms
Myeloproliferative neoplasms (MPNs) commonly result from the acquisition of somatic mutations affecting the Janus kinase 2 (JAK2) (such as JAK2V617F) or the multi-functional protein CALR (such as CALRIns5 and CALRDel52), causing the constitutive activation of signal transducer and activator of transcription (STAT) proteins. The selective oestrogen receptor modulator (SERM) tamoxifen (broadly used in ER+ breast cancer) was effective on eradiating JAK2V617F+ haematopoietic stem and progenitor cells (HSPCs) in preclinical mouse MPN models, which prompted a Phase-II clinical study (Effects of TAMoxifen on the Mutant Allele Burden and Disease Course in Patients with MyeloprolifeRatIve Neoplasms, TAMARIN). In the TAMARIN study, the safety and activity of tamoxifen on reducing mutant allele burden was observed in a subset of MPN patients. This thesis investigated the mechanism of action of tamoxifen in human MPN. According to the transcriptomic analysis, HSPCs from patients meeting the primary or secondary endpoints (tamoxifen responders) were highly enriched with specific molecular pathways such as integrated stress response (ISR), proinflammatory signalling, mitochondrial activity and proteotoxic stress at baseline, implying a predictable transcriptomic signature for tamoxifen susceptivity. Moreover, immunoblotting showed that tamoxifen treatment induced a proapoptotic ISR in tamoxifen–sensitive JAK2V617F+ human cell lines, which manifested as increased translation of activating transcription factor 4 (ATF4) and phosphorylation of eukaryotic initiation factor-2α (eIF2α). The knockdown of ATF4 and inhibition of eIF2α phosphorylation partially rescued tamoxifen-induced apoptosis. Despite the oestrogenic transcriptional regulation found in HSPCs from responders or JAK2V617F+ human cell lines, full-length oestrogen receptors α (ERα), the canonical transcriptional receptor of tamoxifen in mouse haematopoietic system, was not highly expressed in these cells, and the expression of the alternative receptors ERβ and GPER was lower in HSPCs from responders compared with non-responders. Searching for alternative candidate receptors, aryl hydrocarbon receptor (AHR) was found to be highly expressed in responder HSPCs, and its transcriptional activity was activated after tamoxifen treatment. On JAK2V617F+ human cell lines, tamoxifen incubation induced the nuclear translocation and transcriptional activity of AHR, the knock-out of which protected cells from tamoxifen- induced apoptosis, indicating the activation of AHR contributes to its proapoptotic mechanism on tamoxifen-sensitive HSPCs and MPN cell lines. Interestingly, longitudinal transcriptomic analysis of HSPCs from study subjects showed a high baseline expression of OXPHOS-related genes and their specific reduction after 24-week tamoxifen treatment in tamoxifen responders, compared with non-responders, suggesting that tamoxifen exerts a metabolic inhibition on OXPHOS in responder HSPCs. The suppression of cellular respiration and energy synthesis was reproduced in tamoxifen-sensitive JAK2V617F+ human cell lines, bone marrow lineage-negative HSPCs from JAK2V617F+-driven MPN mouse model and patient peripheral blood mononuclear cells (PBMCs), where 4-hydroxytamoxifen (4OH-TAM) directly inhibited mitochondrial respiratory complex I and decreased adenosine triphosphate (ATP) synthesis. This reduction of intracellular ATP significantly suppressed the cytokine-induced pathogenic activation of JAK2 and STAT5 in JAK2V617F+ human cell lines, which was rescued by the respiratory complex II activation and ATP replenishment, explaining the high selectivity of tamoxifen on the JAK-STAT pathway of JAK2V617F+ human cell lines and HSPCs. Overall, tamoxifen exhibits multiple mechanisms in inducing apoptosis of JAK2V617F+ human haematopoietic cells with specific metabolic vulnerabilities (e.g. high OXPHOS dependency and energy requirement) through a combination of transcriptional regulation and metabolic inhibition.
haematopoietic stem and progenitor cells, JAK2V617F, myeloproliferative neoplasms, tamoxifen
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