Characterising the novel MYB-TYK2 fusion gene in high-risk acute lymphoblastic leukaemia: oncogenic potential, effective therapeutic strategies and in vitro modelling of drug resistance mechanisms

Abstract

A growing number of TYK2 activating alterations have been detected in acute lymphoblastic leukaemia (ALL) patients, including the MYB-TYK2 fusion gene. MYB-TYK2 was identified in a high risk (HR) Ph- ALL case and is associated with poor outcome. The transforming and leukaemogenic potential of activating TYK2 alterations, including those of MYB-TYK2, requires clarification in the ALL setting. Tyk2 is a member of the JAK family of tyrosine kinases and similar to JAK fusions, may result in activation of JAK/STAT signalling, and potentially be amenable to JAK inhibitor (JAKi) therapy. In depth characterisation of the underlying mechanisms resulting in leukaemogenesis are warranted and will inform drug response and targetability potential. Moreover, since the development of clinical resistance to targeted therapies such as JAKi, broad investigation of effective small molecule inhibitors (SMIs) is essential. Exploration of the possible modes of drug resistance following long-term treatment and importantly, how to circumvent resistance development, will provide alternative therapies for JAKi resistance in TYK2-altered patients. Hence, this study aimed to 1) identify and characterise the mechanisms of oncogenicity and activated downstream signalling pathways in MYB-TYK2 harbouring cells, 2) assess the efficacy of targeted therapeutics against MYB-TYK2 altered disease and 3) determine the possible mechanisms of resistance to candidate therapy via in vitro models to inform alternative therapeutic approaches to overcome possible disease persistence and resistance. In vitro and in vivo models were used to comprehensively model MYB-TYK2-altered disease. Depending on the downstream experiments, either cytokine-dependent Ba/F3 pro-B cells, Arf-/- pre-B primary mouse cells or NIH-3T3 cells, were retrovirally transduced with the MYB-TYK2 fusion gene isolated from an ALL patient. The targetability of the MYB-TYK2 fusion gene was investigated via high throughput screen (HTS) of 3088 cytotoxic and targeted compounds with further validation of candidates in pre-clinical models. Resistance modelling of the candidate JAKi cerdulatinib was achieved by gradual exposure of cells expressing the MYB-TYK2 fusion gene to increasing concentrations of cerdulatinib over several months. This study elucidated the transformative ability and functional significance of the MYB-TYK2 fusion gene to induce B-ALL in vivo. Results demonstrated the constitutive activation of JAK/STAT signalling due to expression of the MYB-TYK2 fusion gene. HTS identified the HDACi, vorinostat and the HSP90i, tanespimycin as effective targeted therapeutics against cells harbouring the MYB-TYK2 fusion gene in vitro. In addition, the sensitivity of the MYB-TYK2 fusion gene was established to the novel JAKi, cerdulatinib. Both vorinostat and cerdulatinib demonstrated anti-leukaemic effects in pre-clinical in vivo models of the MYB-TYK2 altered disease, resulting in a significantly reduced leukaemic burden. Further investigations into resistance following long-term exposure to cerdulatinib, indicated the likelihood of resistance to cerdulatinib therapy. Results suggested a persistent JAK/STAT activation despite Tyk2 inhibition via possible heterodimer formation with Jak1 and thus, Myb-Tyk2 trans-phosphorylation. This persistent signalling, however, was successfully reversed by HDACi treatment. Overall, the findings presented in this thesis demonstrate, for the first time, the driving potential of TYK2 activating alterations. Through robust modelling and rigorous testing of effective SMIs, this study identified novel therapeutic strategies using vorinostat and cerdulatinib against MYB-TYK2-altered disease. These results provide strong evidence for re-purposing of these drugs as an addition to a chemotherapy backbone for treatment of this highly aggressive subtype of B-ALL. Further insights into the resistance profile in response to cerdulatinib, as well as incorporation of targeted therapeutics to overcome this resistance, will assist high-risk patients in a clinical setting. These findings contribute to precision medicine approaches and will ultimately improve outcome and long-term survival for patients harbouring activating TYK2 alterations.