Overcoming Ibrutinib and Venetoclax resistance in Chronic Lymphocytic Leukaemia.

慢性淋巴细胞白血病（CLL）是一种血癌，影响帮助抵抗感染的B细胞。它是西方世界最常见的白血病，CLL患者死于感染的风险很高。伊布替尼（BTK抑制剂）和维奈托克（BCL 2抑制剂）已经彻底改变了CLL的治疗方法，但它们不能治愈，一些患者会产生耐药性。在个性化医疗时代，挑战在于为正确的患者确定正确的药物。这将确保患者获得最佳治疗，同时降低与治疗失败相关的医疗费用。CLL细胞的存活和扩增依赖于它们在外周血和淋巴结之间迁移的能力。我们和其他人已经确定，伊曲替尼单独或与维奈托克联合使用，可以使一些患者的CLL细胞通过不同的途径（TLR9途径）发出信号，导致迁移和耐药性增加。我们已经开发了一种TLR9反应测定法来前瞻性地识别这些患者。这种简单的迁移试验鉴定了三组患者：TLR9-应答者（在没有任何药物的情况下通过TLR9发出信号）、TLR9-致敏者（在存在伊曲替尼的情况下仅通过TLR9发出信号）和TLR9-非应答者（从不通过TLR9发出信号）。我们认为，TLR9应答组和TLR9致敏组都可能产生耐药性，并将受益于TLR9抑制策略与含伊曲替尼的治疗的组合。相反，TLR无应答者代表了一组最有可能对含伊匹替尼治疗产生最佳应答而无需其他靶向干预的患者。本申请的目的1是在大型患者队列中验证该测定。这种检测方法的成功验证和临床采用将使患者能够避免压力，副作用和昂贵的治疗，他们不会响应虚假的希望。为此，我们将在200份来自继续接受伊鲁替尼单药或维奈托克治疗的初治患者的样本中测试我们的检测方法的预测价值。这些样品来自UK-CLL FLAIR试验，我们不仅可以获得这些样品，还可以获得治疗后患者的临床结果（无进展生存期）和反应（最小残留疾病状态）。本申请的目的2是使用来自TLR9应答者和TLR9致敏病例的CLL细胞来鉴定新的药物靶标，其可以潜在地阻断TLR9介导的耐药性。我们最初的重点将是NF-kB途径基因，因为BTK，BCL 2和TLR 9会聚在该途径上，并且它是CLL细胞迁移和存活的已知驱动因素。在初步研究中，我们已经通过TLR9激活了原代CLL细胞，并鉴定了不同的NF-κ B特征，其与上述不同的TLR9应答类别相关。对此进行更深入的研究将有助于我们将不同的迁移反应与不同的NF-κ B信号联系起来，从而导致基因转录的特定变化。反过来，这应该使我们能够确定有希望的TLR9反应组特异性药物靶点，这些靶点将克服对当前疗法的耐药性。这第二个目标将使用所描述的实验室实验和机械数学建模来实现。数学建模方法已被广泛用于破译NF-kB信号传导的复杂性，它们产生的有希望的靶标将在实验室实验中进行测试，以确定它们是否可以逆转耐药性。我们有一个正在进行的合作，CRUK资助的药物发现团队将为我们提供新的NF-κ B信号通路抑制剂，以测试一些这些模型预测的目标。因此，希望这个项目将验证伊鲁替尼/维奈托克的反应预测器，并确定有希望的耐药患者的治疗策略。

Chronic Lymphocytic Leukaemia (CLL) is a blood cancer affecting cells that help fight infection called B cells. It is the commonest Leukaemia in the western world and patients with CLL have a high risk of dying from infections. Ibrutinib (BTK inhibitor) and venetoclax (BCL2 inhibitor) and have revolutionised treatment of CLL but they are not curative and some patients develop resistance. In the era of personalised medicine, the challenge is to identify the right drugs for the right patients. This will ensure that patients get the best treatments whilst reducing healthcare costs associated with treatment failure. Survival and expansion of CLL cells are dependent on their ability to migrate between the peripheral blood and lymph nodes. We, and others, have identified that ibrutinib, alone or in combination with venetoclax, primes CLL cells, from some patients, to signalling through a different pathway, the TLR9 pathway, resulting in increased migration and drug-resistance. We have developed a TLR9-response assay to prospectively identify these patients. This simple migration assay identifies three groups of patients: TLR9-responders (signal through TLR9 in the absence of any drugs), TLR9-sensitised (only signal through TLR9 in the presence of ibrutinib) and TLR9-non-responders (never signal through TLR9). We believe that both the TLR9-responder and TLR9-sensitised groups are likely to develop drug-resistance and would benefit from a TLR9 inhibition strategy in combination with ibrutinib-containing treatments. Conversely, the TLR-non-responders represent a group of patients most likely to have an optimal response to ibrutinib-containing treatments without the need for other targeted intervention. Aim 1 of this application is to validate this assay in a large cohort of patients. Successful validation and clinical adoption of this assay will enable patients to avoid the stress, side effects and false hope of expensive therapies that they won't respond to. To do this we will test the predictive value of our assay in 200 samples from treatment naïve patients who went on to be treated with ibrutinib alone or with venetoclax. The samples are from the UK-CLL FLAIR trial and we have access not only to these samples, but also to the clinical outcome (progression-free survival) and response (minimal residual disease status) of patients following treatment.Aim 2 of this application is to use CLL cells from the TLR9-responder and TLR9-sensitised cases to identify novel drug targets, which could potentially block TLR9-mediated drug-resistance. Our initial focus will be on NF-kB pathway genes as BTK, BCL2 and TLR9 converge on this pathway and it is a known driver of CLL cell migration and survival. In preliminary studies we have activated primary CLL cells through TLR9 and identified different NF-kB signatures, which correlate with the different TLR9-response categories described above. More in-depth studies of this will help us to link the different migratory responses with distinct NF-kB signalling, resulting in specific changes in gene transcription. In turn, this should enable us to identify promising TLR9-response group-specific druggable targets that will overcome resistance to current therapies. This second aim will be achieved using both the laboratory experiments described and mechanistic mathematical modelling. Mathematical modelling approaches have been extensively used to decipher the intricacies of NF-kB signalling and the promising targets that they generate will be tested in laboratory experiments to establish if they can reverse drug resistance. We have an on-going collaboration a CRUK-funded drug discovery team who will provide us with novel NF-kB signalling pathway inhibitors in order to test some of these model-predicted targets.Therefore, it is hoped that this project will validate a response predictor for ibrutinib/venetoclax and also identify promising therapeutic strategies for drug-resistant patients.