The introduction of tyrosine kinase inhibitors in chronic myeloid leukaemia (CML) has revolutionised disease outcome. However, despite this, progression to blast phase disease is high in those that do not achieve complete cytogenetic and major molecular response on standard therapy. As well as BCR-ABL-dependent mechanisms, disease persistence has been shown to play a key role. Disease persistence suggests that, despite a targeted therapeutic approach, BCR-ABL-independent mechanisms are being exploited to sustain the survival of a small population of cells termed leukaemic stem cells (LSCs). Increasing evidence highlights the importance of self-renewal and survival pathways in this process. This review will focus on the role of stem-cell restricted self-renewal pathways, namely Hedgehog, Notch, and Bone Morphogenic Pathway (BMP). Wingless-Int/β-Catenin (Wnt/β-Catenin) signalling will be discussed within a further review in this series in view of its regulatory role in GSK3β. Further to this, we will highlight the role of key transcriptional regulators, namely p53 and c- MYC, in targeting wider deregulated networks. Keywords: Self-renewal, signalling, CML, leukaemic stem cells.
Background: The BCR‐ABL1 chimeric oncoprotein drives chronic myeloid leukaemia (CML) pathogenesis. The introduction of tyrosine kinase inhibitors (TKIs) has transformed clinical outcomes for patients with CML, with over 80% of those treated with imatinib (IM) surviving for more than 10 years. Second and third‐generation TKIs are more potent and can impede the emergence of resistance, inducing deep molecular responses in both untreated and IM‐resistant CML patients. Despite the success of TKIs, up to 35% of patients discontinue their TKI due to intolerance or develop resistance and this remains a key area of unmet clinical need. Asciminib (formerly ABL001) is a potent and selective allosteric inhibitor of ABL1 that binds to the myristoyl binding pocket of ABL1 to hold BCR‐ABL1 in an inactive conformation. Preclinical studies have shown that asciminib selectively inhibits the growth of BCR‐ABL1 positive (+) cells regardless of the presence of BCR‐ABL1 point mutations 1 . Clinical trials for patients with CML or Ph+ acute lymphoblastic leukaemia, testing asciminib alone and in combination with TKIs are currently underway and preliminary results are promising. Aims: To assess if dual inhibition of BCR‐ABL1 leads to improved treatment outcomes in preclinical studies. Methods: Here, we assess the effects of asciminib, alone and in combination with ATP‐competitive TKIs (IM, nilotinib (NIL) and ponatinib (PON)) in CML cell lines and primary CD34+ chronic phase (CP)‐CML stem/progenitor cells (HSPC). We assessed synergy using resazurin readouts using CompuSyn Software. We performed cell counts, apoptosis, cell‐cycle and proliferation assays to determine the effect of drug combinations in CML cell lines and primary samples; and confirmed effects on primitive cells using colony‐forming cell (CFC) and long‐term culture‐initiating cell (LTC‐IC) assays in vitro . Results: Dose‐response studies using the resazurin assays in CML cell lines (Bv173, K562, KCL22) indicate that asciminib is potent at low nanomolar concentration, even in cells that express the BCR‐ABL1 T315I point mutation (KCL22). Apoptosis and cell cycle assays’ assessed by FACS showed that the inhibitory effects of asciminib were maintained in KCL22 T315I ‐expressing cells when asciminib was used in combination with PON. Washout studies with asciminib in KCL22 WT/T315I ‐expressing cells demonstrated a prolonged phenotypic response using low‐nanomolar doses of asciminib as the cells failed to regrow and had irreversible cell‐cycle damage. Primary CD34+ CML HSPCs demonstrated proliferation arrest and increased apoptosis (70–100% increase relative to control; p < 0.001) when treated for up to 72 hours with asciminib, alone and in combination with IM or NIL. LTC‐IC and CFC assays, determining the functional activity of primitive CML HSPCs in vitro, demonstrated that the combination of asciminib with IM or NIL reduced colony outputs (60–90% decrease relative to controll, p < 0.001 ) , beyond that achieved with each drug alone (40–80% relative to control, p < 0.001 ) , and in separate experiments, with minimal effect on normal HSPC. Summary/Conclusion: These results suggest that asciminib represents a novel therapeutic approach with effects on primitive CP‐CML HSPCs both as a single agent and in combination with TKI and has efficacy in cells expressing the multi‐TKI resistant T315I mutation. We are now investigating the mechanism of action for asciminib, alone and in combination with NIL, by RNA‐sequencing.
We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.
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