Overcoming Ibrutinib and Venetoclax resistance in Chronic Lymphocytic Leukaemia.

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.

Inhibitors of BTK and BCL2 have revolutionised the treatment of CLL but they are non-curative, expensive and some patients develop resistance. Resistance has been linked with the clonal expansion of CLL cells exhibiting BTK and BCL2 mutations. However, these mutations are usually only evident in a minor subset of the tumour and in a small percentage of resistant patients. Therefore, other explanations are required for the majority of cases who only partially respond or become refractory to treatment. We, and others, have shown that ibrutinib, both alone and with venetoclax, primes CLL cells from some patients to signalling through the TLR9 pathway inducing drug-resistance. We have developed a TLR9-response migration assay with the capacity to prospectively identify these patients. The aims of this application are: (1) To test our TLR9-response assay's ability to predict for clinical outcome and MRD status in 200 treatment naïve samples from the UK-CLL FLAIR trial. If successful, this would be a simple predictive tool to identify ibrutinib/venetoclax resistant patients. (2) To use samples from these resistant patients to identify novel druggable targets that block TLR9-mediated resistance. Initially, we will focus on NF-kB as BTK, BCL2 and TLR9 converge on this pathway and preliminary data from our TLR9-resonse assay defines CLL groups with different NF-kB signalling. However, we will also consider unexpected routes to resistance using systems biology approaches. (3) Establish the best drug targets using mathematical simulations before confirming their potential in 'wet lab' experiments. We have an on-going collaboration a CRUK-funded drug discovery team who will provide us with novel NF-kB signalling pathway inhibitors to test some of these model-predicted targets. Therefore, this project should predict patient responses to ibrutinib/venetoclax and identify a novel therapy for drug-resistant patients. Insights gained will be applicable to other B-cell malignancies.