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The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common γ chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations.

Acute lymphoblastic leukemia (ALL), which is the most common cancer in children, shows extensive genetic intra-tumoral heterogeneity. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. In order to envision the clinical implementation of a refined risk-category strategy based on ALL subclonal composition, it is essential to first generate a reference single-cell map and accumulate evidence on how the subclonal composition affects the response to treatment. For that, we performed large-scale and integrative single-cell genome and transcriptome profiling of pediatric samples at diagnosis, during drug treatment and in case of relapse. We used the 10x Genomics platform for single-cell RNA-sequencing analysis (around 4000 cells per sample) and the Tapestri Platform (Mission Bio) for targeted single-cell DNA-sequencing (around 5000 cells per sample) of the most mutated genomic regions in ALL. For the later, we developed a custom panel that covers 305 ALL mutational hotspots across 110 genes. We have determined a reference single-cell map of the cellular (based on the gene expression profile) and the clonal composition (based on the co-occurrence of mutations at each individual cell) for pediatric ALL at diagnosis (8 T-ALL and 10 B-ALL patients). We have also reconstructed the tumor phylogeny highlighting the order of mutational acquisition and the most likely pattern of evolution. Moreover, we have studied how T-ALL evolves during drug treatment at single-cell resolution in 4 patients, unraveling which are the most sensitive clones to the therapy, which are the most resistant ones and when relapse clones originated. Single-cell RNA-sequencing also provided information on how normal hematopoiesis recovers during chemotherapy treatment. The results show that ALL is typically composed by a major clone and 5-10 smaller clones that have different sensitivities to the therapy. We have been able to detect minor clones ( Disclosures Maertens: Gilead Sciences: Other: Grants, personal fees and non-financial support; Cidara: Other: Personal fees and non-financial support; Amplyx: Other: Personal fees and non-financial support; F2G: Other: Personal fees and non-financial support; Merck: Other: Personal fees and non-financial support; Pfizer: Other: Grant and personal fees; Astellas Pharma: Other: Personal fees and non-financial support.

CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the Fip1l1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line. Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones. Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes Fip1l1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in Fip1l1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of Fip1l1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of Fip1l1 and dependent on the interruption of the juxtamembrane region of PDGFRA. The expression and phosphorylation levels of Fip1l1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous Fip1l1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA. The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting. Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene. In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

Activation mutations in JAK3 occur in 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases, and co-occur frequently with HOXA cluster rearrangement. Genomic rearrangement of the HOXA cluster results in increased expression of HOXA9 and HOXA10. However it remains unclear if either HOXA9 or HOXA10 can cooperate with activating JAK3 mutations during oncogenic transformation and leukemogenesis. We have previously shown that JAK3 mutations lead to cell transformation and cause a long latency T-ALL in vivo using a mouse bone marrow transplant model. In this study we demonstrate that co-expression of the activating JAK3(M511I) protein with HOXA9 cooperate to develop leukemia within 30 days of transplant using an in vivo bone marrow transplant model. In our cooperative model, murine hematopoietic stem / progenitor cells were co-transduced with either both retroviral vectors encoding JAK3(M511I)/GFP and HOXA9/mCherry or each individually and then injected into sub-lethally irradiated recipient mice. Mice transplanted with bone marrow cells expressing JAK3(M511I) mutant alone developed T-ALL in 120 to 150 days. In sharp contrast, mice transplanted with cells expressing both JAK3(M511I) and HOXA9 showed rapid leukemia development within 30 days after transplant. Leukemia development was characterized by the rapid and specific increase in GFP-mCherry double positive cells. These animals showed high WBC, and splenomegaly and accumulation of immature CD8 single positive cells in the thymus. Similar experiments with HOXA10 did not show cooperation suggesting that HOXA9 is the more important oncogene in HOXA rearranged leukemias when a JAK3 activating mutation is present. To determine the underlying genetic mechanism for cooperation between HOXA9 and JAK3(M511I) the single positive JAK3 and double positive JAK3/HOXA9 expressing cells were isolated from thymi of leukemic mice for both epigenomic profiling using ATAC-seq and gene expression profiling. These analyses identified genetic pathways activated by the co-expression of HOXA9 and JAK3(M511I) mutation and provide mechanistic insight into the synergistic interaction between these two factors in driving leukemia development. Treatment of the animals with a JAK kinase inhibitor resulted in delayed leukemia development, confirming that the leukemia cells remain sensitive to the JAK inhibitor. This mouse model provides insight in the cooperation between oncogenes in leukemia development and provides a model for the study of targeted agents in this setting. Disclosures No relevant conflicts of interest to declare.