Unraveling the Molecular Signature of BCR-ABLP210 and BCR-ABLT315I in the Drosophila Melanogaster CML Model

Abstract

Background: Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm characterized by the Philadelphia chromosome, a reciprocal t (9;22) chromosomal translocation leading to the fusion of the BCR gene on chromosome 22 and the ABL gene on chromosome 9, and resulting in the formation of the BCR-ABL1 fusion gene. This gene encodes for the BCR-ABL1 oncoprotein that drives the uncontrolled cell proliferation via its constitutively active tyrosine kinase domain in the ABL1 region. Tyrosine kinase inhibitors (TKIs) were established as targeted therapies and are currently the standard first-line treatment for patients with CML. However, BCR-ABL1 mutations have been a major mechanism of resistance. Patients with BCR-ABLT315I gatekeeper point mutation, a Threonine to Isoleucine substitution at position 315 in the ATP-binding region of the ABL1 kinase domain, are resistant to first and second generation TKIs. Currently, the only FDA approved drugs which patients with this mutation respond to are the third generation TKI, ponatinib, and the first allosteric inhibitor, asciminib. However, these drugs have severe side effects which affect compliance and their efficacy is majorly in chronic phase CML patients. Therefore, the main aim of this study is to find novel potential targets against BCR-ABLT315I to aid in this unmet clinical need. However, our approach will allow us to identify common targets against BCR-ABLp210 and BCR-ABLT315I mutant should they arise.

Methods: We will express BCR-ABLp210 and BCR-ABLT315I in the hemolymph system of the Drosophila melanogaster utilizing the Hml Δ driver to test if their expression in this system will result in a phenotype be it an increased hemocyte count, disruption in the sessile hemocyte banding pattern, or a dysregulation in the humoral arm of the innate immunity. The effect on the innate immunity will be studied via RT-PCR quantification of downstream AMPs of the Toll, Imd, and JAK/STAT pathway namely Drosomycin, Diptericin A, and TotA in the hemolymph, respectively. As such, hemolymph will be collected from third-instar larva, RNA will be extracted, cDNA will be synthesized, then RT-PCR will be performed. We will also collect hemolymph for RNA sequencing in order to determine differentially expressed genes (DEGs) between groups to identify potential targets. As such, Hml Δ-Gal4; UAS-GFP flies will be crossed with w1118 (wt), UAS-BCR-ABLP210, and UAS-BCR-ABLT315I flies for these experiments. For the RNA sequencing, we will utilize the Illumina NovaSeq6000 and perform RNA sequencing with a 100bp paired-end read and 40 million reads per sample. We will also generate screening lines harboring the Hml Δ driver and the BCR-ABLp210 and BCR-ABLT315I transgenes which will be validated by immunofluorescence since the BCR-ABL1 genes are tagged with Myc as a reporter. DEGs will be cross-referenced with transcriptome of CML patients and those patients with the BCR-ABLT315I mutation specifically if available. Selected genes will be validated via RT-PCR. In case we identify upregulated screening genes, then we will cross our screening lines with flies harboring the RNAi system specifically for the target genes. Then we will check hemocyte count and banding pattern to check for partial or total rescue of the phenotype.

Results: Upon the expression of BCR-ABLp210 and BCR-ABLT315I, there was an increase in hemocyte count and a disruption in the sessile banding pattern. An exacerbated version of the phenotypes was noted in the BCR-ABLT315I mutant. The hemocyte count in BCR-ABLp210 flies was significantly higher than the control and the BCR-ABLT315I flies had an even higher hemocyte count as compared to BCR-ABLp210 flies. Moreover, the disruption in the sessile pattern was partial in the BCR-ABLp210 flies when compared to the control while it was total in the BCR-ABLT315I flies. Furthermore, we identified dysregulation in the Toll, Imd, and JAK/STAT pathways at the mRNA level in both the 3rd instar larva and adult stage via measuring, Drosomycin, Diptericin A, and TotA, respectively. Transcriptomic analysis showed us similar molecular signatures for BCR-ABLp210 and BCR-ABLT315I but they were very distinctive than that of the wt control. After cross-referencing upregulated genes against adult, pediatric CML patient samples and BCR-ABLT315I mutant mice, a total of six genes were chosen (Rapgap1, CG9265, meltrin, sprint (spri), wing blister (wb), and zasp52). Knockdown of Rapgap1 partially rescued the hemocyte count in BCR-ABLp210 and BCR-ABLT315I flies. Knockdown of spri and CG9265 partially rescued the hemocyte count in BCR-ABLT315I flies whereby their count decreased to levels comparable to the less aggressive wild type BCR-ABLp210. However, knockdown of spri increased the hemocyte count in BCR-ABLp210 flies. Moreover, knockdown of meltrin partially rescued the sessile hemocyte banding pattern in BCR-ABLp210 and BCR-ABLT315I flies; however, it increased their hemocyte counts so it also had an oncogenic effect. wb RNAi increased the hemocyte count in BCR-ABLP210 and BCR-ABLT315I flies while spri RNAi and zasp52 RNAi had no effect.

Conclusion: We expressed BCR-ABLP210 and BCR-ABLT315I in Drosophila melanogaster hematopoietic system that resulted in phenotypic consequences such as increasing circulating hemocyte count and disruption in the sessile hemocytes’ banding pattern. In addition, their expression dysregulated the humoral Toll, Imd, and JAK/STAT pathways at the mRNA level in both the 3rd instar larva and adult stage. The BCR-ABLT315I mutant demonstrated a more complex oncogenic effect than that of the wild type BCR-ABLP210 by presenting with more severe phenotypes and a higher deviation in humoral dysregulation. Furthermore, we unraveled the transcriptome of our hematopoietic BCR-ABLP210 and BCR-ABLT315I CML Drosophila models whereby their molecular signatures were very similar to each other but distinctly different than that of wt control. In addition, we identified four potential target genes that had a rescue effect on both the BCR-ABLp210 and BCR-ABLT315I flies namely, Rapgap1, spri, CG9265, and meltrin. The validity of this hematopoietic CML model is strengthened by the fact that upon cross-referencing our genes with adult, pediatric CML patient samples, and BCR-ABLT315I mouse model, several genes were found to be differentially expressed when compared to wt control and they were differentially expressed in the same direction be it over or under expressed. The novelty in this study lies in the power of this hematopoietic Drosophila CML model. It allowed us to target some genes that were not highlighted before in the literature pertaining to leukemia. We have identified targets that are common to both BCR-ABLp210 and BCR-ABLT315I which are worth pursuing since CML patients with and without the T315I mutation can benefit alike. There are still more upregulated genes to be screened for in the pipeline and many more taking into account the down regulated genes.