Using in vivo modelling to investigate therapeutic approaches to reverse/prevent disease resistance in children with high-risk Ph-like B-ALL and T-ALL treated with targeted therapies.
Dr Laura Eadie
Relapsed acute lymphoblastic leukaemia (ALL) is the leading cause of childhood non-traumatic death (15% of T-ALL and 20% of B-ALL patients relapse). Chemotherapy results in adverse side effects and a lifelong risk of other malignancies. Risk stratification and targeted therapy based on the molecular genetics of an individual’s disease is warranted. We will test the efficacy of novel drugs and combination therapies in mouse models of ALL. Findings will inform clinical practice; therapeutic strategies will be optimised to ensure the best chance of cure for children with high-risk forms of ALL.
Laura Eadie, Deborah White
Relapsed Acute Lymphoblastic Leukaemia (ALL) is a significant medical problem in children and recent advances in genomic profiling techniques have highlighted the genetic complexity of the disease. Patient derived xenografts of ALL are the gold standard for mimicking human disease in laboratory models. This project has successfully generated mouse avatars for 15 high-risk ALL patients (with a further 5 patient avatars ongoing). These avatars will be used to model drug resistance and test efficacy of new therapeutic strategies compared with the current standard of care.
Ultimately, the outcomes of my research will provide clinicians with an arsenal of effective potential targeted therapies for Australians with high-risk ALL. These studies will contribute to our ability to offer precision medicine to patients, transforming the future of treatment and care for Australian children with ALL.
Acute Lymphoblastic Leukaemia (ALL) is a malignant disorder of lymphoid progenitor cells and is divided into two major immunophenotypic groups: B cell precursor ALL (B-ALL, ~80% of ALL) and T-cell ALL (T-ALL, ~20% of ALL). Relapsed ALL is a significant medical problem in children and recent advances in genomic profiling techniques have highlighted the genetic heterogeneity of the disease. Of importance is the high-risk ALL subtype Ph-like ALL. This disease typically responds poorly to current chemotherapeutic regimens but is targetable with safe and highly potent drugs already in clinical use. This project uses mouse models to recapitulate human disease and investigate efficacy of novel and combination treatment strategies and consists of two aims.
Aim 1 – establish in vivo models (PDX) of resistance using primary cells from children with Ph-like ALL and TALL
I am the first person at SAHMRI to establish and optimise xenograft models from whole-body irradiated mice. During the term of this grant, I have autonomously generated primary patient derived xenografts (PDX) for seventeen ALL patients. Primary engraftments expand the leukaemic cell population by using immune-compromised mice as patient avatars. Recapitulation of human disease in a subset of these engraftments has been verified by next generation sequencing confirming the validity of my disease model.
Engraftment of human cells is monitored by routine sampling of blood and flow cytometric analysis; mice are humanely killed and mononuclear (target) cells isolated once engraftment is >50%. I currently have cryopreserved cells from nine B-ALL patients, six T-ALL patients and two mixed phenotype ALL (MPAL) patients encompassing a range of high-risk genomic alterations including:
- NUP214-ABL fusion (B-ALL patient)
- NUP214-ABL1 fusion (T-ALL patient)
- NUP214-SET1 fusion (T-ALL patient)
- PAX5-SRCIN1 fusion (B-ALL patient, novel alteration)
- ETV6-ABL1 fusion (B-ALL patient)
- P2RY8-CRLF2 fusion (2x B-ALL patients)
- A number of patients harbouring nonsynonymous mutations to genes such as CDKN2a, NRAS, NOTCH2, KMT2D
I have a further five primary PDX models which are ongoing as the level of engraftment is currently <50%. The first secondary engraftment and evaluation of a novel treatment strategy will be performed in later 2019.
Of interest, a recent B-ALL PDX model of the high-risk ETV6-ABL1 fusion resulted in leukaemic engraftment within the brain, indicative of central nervous system (CNS) involvement. Prognosis for ETV6-ABL1 disease is typically poor and this PDX model suggests an aggressive disease. CNS penetration in ALL is a significant un-met clinical need, particularly prominent during disease relapse. Two additional PDX models I established in early 2018 (from patients with early T-cell precursor ALL and T-ALL) resulted in rapid detection of leukaemic cells within the blood (>95%) but not in the bone marrow or brain. These results highlight the complexity of individual leukaemias and the need for real-time disease imaging and tracking. These preliminary results form the basis of ongoing studies where fluorescently-tagged cells harvested from PDX models will be used to track disease dissemination and investigate CNS involvement (this work is funded by the SAHMRI Precision Medicine Theme 2019-2021).
Aim 2 – determine the efficacy of targeted therapeutic approaches in PDX models developed in Aim 1.
Two Ph-like ALL fusions were investigated in in vitro models of disease (NUP214-ABL and SNX2-ABL1 fusions) to determine the sensitivity of these gene fusions to a novel therapeutic agent. ABL1 fusions can be targeted with tyrosine kinase inhibitors (TKIs) such as dasatinib, imatinib and nilotinib, which are used to treat patients with chronic myeloid leukaemia (CML). There is also a new allosteric inhibitor called asciminib that has demonstrated efficacy against the BCR-ABL1 fusion most commonly observed in CML patients. The efficacy of asciminib against other ABL1 fusions has not previously been tested and was the focus of this body of work.
This section of my project used the innovative Invitrogen Gateway® cloning system (I established and optimized this system in our laboratory) to facilitate the expression of fluorescently labelled, PCR-amplified DNA from patient cells. Empty vector equivalents were used as control. Viral constructs containing fusion genes of interest were created for transduction of the B-ALL cell line, Ba/F3. Ba/F3 cells stably expressing the fusion genes of interest were then used to ascertain the efficacy of asciminib both alone and in combination with either imatinib, nilotinib or dasatinib.
Cell death assays were conducted to determine the asciminib LD50, the concentration of asciminib required to kill 50% of Ba/F3 SNX2-ABL1 cells. Results demonstrated that when used as a monotreatment, asciminib was unable to decrease cell viability at clinically achievable concentrations. However, when asciminib was used in combination with either imatinib, nilotinib or dasatinib, the amount of asciminib required to achieve 50% cell death was significantly lowered. Importantly, the concentration of TKI used in the combination experiments had no significant effect on cell death when used alone indicating combination therapy was required for effective Ba/F3 SNX2-ABL1 cell death. Conversely, when asciminib was used as a monotreatment against Ba/F3 NUP214-ABL1 cells, viability was significantly decreased at the clinically achievable concentration of 5 mM. Investigations into asciminib as a combination therapy are ongoing.
These results demonstrate that while asciminib is unlikely to be an effective monotherapy for patients harbouring the SNX2-ABL1 gene fusion, it may be efficacious when used in combination with a TKI. Using drugs in combination is an attractive therapy option since sub-efficacious doses of multiple drugs induces fewer off target side effects while still resulting in leukaemic cell death. These results also demonstrate that different ABL1 gene fusions respond to asciminib treatment differently highlighting the complexity of drug:target interactions. Results from this aim will be used to guide drug treatment strategies in in vivo mouse models. Importantly, the primary PDX models for patients harbouring a range of ABL1 gene fusions have already been completed facilitating rapid establishment of secondary PDX and drug efficacy evaluation.
There have been no publications resulting from this work to date.
I am an SA Cancer Council Beat Cancer Project Mid-Career Fellow (2019-2021) and the results from this project formed the preliminary data used in my successful application for this fellowship.