PF170 ETV6/RUNX1 FUSION GENE ABROGATION DECREASE THE ONCOGENIC POTENCIAL OF TUMORS CELLS IN A PRECLINICAL MODEL OF ACUTE LYMPHOBLASTIC LEUKEMIA
Authors: A. Montaño, J.L. Ordóñez, V. Alonso‐Pérez, A. Marín‐Quílez, C. Pérez‐Carretero, R. Benito, I. García‐Tuñón, J.M. Hernández‐Rivas
Journal: HemaSphere (2019)

Background: The gene fusion between ETV6 and RUNX1 generated by t(12;21)(p13; q22), is the most frequent chromosomal translocation in children with acute lymphoblastic leukemia (ALL). Patients carrying this translocation are associated with a good prognosis and excellent molecular response to treatment. However up to 20% of cases relapse. Furthermore, the response to the treatment of some relapse cases is associated by resistance to treatments such as glucocorticoids, and these patients must be treated with stem cell transplantation. Recent studies suggest that ETV6/RUNX1 ( E/R ) plays a role in the initiation of leukemia and is also essential for disease progression and maintenance, through the deregulation different molecular pathways that contribute to leukemogenesis, such as the upregulation of PI3K/AKT/mTOR pathway. Aims: To study the “in vitro” and “in vivo” effects of E/R fusion gene abrogation by using CRISPR/Cas9, on the oncogenic potential of tumor cells (REH cell line). Methods: 1) Based on CRISPR/Cas9 system, sgRNAs directed towards the fusion gene were designed to produce indels modifying the oncogene ORF and, therefore, the expression of the protein. Tumor cells (REH cell line expressing E/R fusion gene) were electroporated (Amaxa nucleofector, Basilea, Switzerland) and sorting by flow cytometry.qPCR and Western Blot were used to checkE/R mRNA expression and downstream targets expression. 3) Cell viability was measured by MTT cell proliferation assays in E/R KO cells and E/R positive cells treated with copanlisib, a PI3K inhibitor, (10 nM) and prednisolone (250 μM). 4) In a xenograft model, E/R positive cells (right flank) and E/R KO cells (left flank) were subcutaneously injected in 16 NOD/SCID/IL2 receptor gamma chain null mice. Excised tumors were sampled just after sacrifice and stained with hematoxilin &amp; eosin. Results: 1) A total loss of E/R expression was observed in different E/R KO clones stablished by single cell, demonstrating an effective disruption of the oncogene in REH cells. Moreover, a decrease of downstream targets expression levels such us, phospho‐Akt (66%), BCL‐XL (48%) and BCL‐2 (52%) was observed. 2) Abrogation of E/R fusion gene showed a significantly decrease of oncogenic potential “in vivo”. Mice injected with E/R KO cells did not generated tumors or generated significantly smaller tumors than those generated by E/R positive cells. Furthermore, a higher rate of mitotic activity was observed in tumors from E/R positive cells (62 vs 20; p = 0.006). 3) Tumor cells showed a higher sensitivity to copanlisib and to combination of copanlisib and prednisolone “in vitro” after E/R depletion. Treatment with copanlisib raised to decrease the cell viability up to 35% in E/R KO cells vs. 55% in E/R positive cells (p &lt; 0,05). In the same way, combination of copanlisib and prednisolone was more effective in E/R KO cells (26 % vs 34% of cell viability, p &lt; 0.05). Summary/Conclusion: Avoiding the E/R fusion gene expression reduces significantly the oncogenic potential of ALL cells (REH, E/R positive) both “in vivo” and “in vitro”. E/R KO cells also showed an increased sensitivity to copanlisib alone and in combination with prednisolone, suggesting E/R expression could be involved in the prednisolone resistance observed in some patients. These results showed that E/R plays an important role in the maintenance of the leukemic phenotype. The fusion gene could therefore become a potential therapeutic target.