PS933 DISCOVERING PATHWAYS INVOLVED IN RESISTANCE TO INDUCTION THERAPY IN T‐ALL TO TREAT REFRACTORY AND RELAPSED DISEASE
Authors: Beckett M. , Shi Y. , Blair H. , Nakjang S. , Tirtakusuma R. , Szoltysek K. , Krippner‐Heidenreich A. , Heidenreich O. , van Delft F.W. 
Published: June 2019

Background:

Survival for acute lymphoblastic leukaemia has increased over the last 50 years giving us around 80% survival in paediatric patients. These good results can be attributed to the refinement and response driven administration of intensive chemotherapy regimens. Despite the overall good survival, for patients with relapsed or refractory disease the outcome remains poor. Vincristine, Dexamethasone, Asparaginase and Daunorubicin (VXLD) form the cornerstones of T‐ALL induction therapy. Response to induction therapy is predictive for outcome. Identifying pathways that contribute to resistance to induction chemotherapy will help identify novel drug targets that could improve the efficacy of treatment for patients with poor response to current therapy.

Aims:

We aim to identify new drug targets in pathways involved in resistance to improve remission rates and outcomes for patients with refractory and relapsed T‐ALL.

Methods:

We use an orthotopic xenograft model of T‐ALL and a four‐drug treatment regimen based upon current T‐ALL therapy to mimic the clinical setting. T‐ALL cells were transduced with a genome wide knock out library prior to long term in vitro culture or intra‐femoral injection into immunocompromised mice. T‐ALL cells were harvested from cultured cells and from murine bone marrow to extract DNA for PCR amplification of guide containing regions. Next generation sequencing was used to determine representation of guides after VXLD treatment. The population of guides of cells retrieved from drug treated cells was compared to baseline samples and vehicle control treated mice in order to ascertain which guides are enriched or depleted by chemotherapeutic pressure.

Results:

We demonstrated the potential of identifying resistance mechanisms through combining the use of a clinically relevant multi agent chemotherapy regimen and concurrent CRISPR screening. Our pilot studies showed T‐ALL induction therapy increases the survival of T‐ALL engrafted mice and reduces leukemic burden. Relevant to the aim of this project, the administration of VXLD treatment resulted in differential sgRNA representation compared with control treatment, allowing us to explore mechanisms of drug resistance to conventional induction chemotherapy. We have already analysed the initial sequencing results which showed good quality reads as well as guide and library coverage. We observed enrichment of sgRNAs targeting the tumour suppressor PTEN in a cell line with wild type PTEN function suggesting that activation of PI3K/AKT/mTOR signalling provides proliferative advantage in this experimental setting. We also identify pathways associated with differentially expressed sgRNAs, including those linked to checkpoints in T‐cell development. Their functional role and suitability as additional drug targets will be explored further.

Summary/Conclusion:

We used a genome wide knock out CRISPR library whilst applying chemotherapeutic pressure in vitro and in vivo conditions to identify pathways which may contribute to drug resistance to T‐ALL induction therapy. Future work will focus on validation of differential sgRNA expression underlying these drug resistance mechanisms and ultimately improve efficacy and reduce toxicity of induction treatment.