Despite incredible progress in cancer treatment, therapy resistance remains the leading limiting factor for long−term survival. During drug treatment, several genes are transcriptionally upregulated to mediate drug tolerance. Using highly variable genes and pharmacogenomic data for acute myeloid leukemia (AML), we developed a drug sensitivity prediction model for the receptor tyrosine kinase inhibitor sorafenib and achieved more than 80% prediction accuracy. Furthermore, by using Shapley additive explanations for determining leading features, we identified AXL as an important feature for drug resistance. Drug−resistant patient samples displayed enrichment of protein kinase C (PKC) signaling, which was also identified in sorafenib−treated FLT3−ITD−dependent AML cell lines by a peptide−based kinase profiling assay. Finally, we show that pharmacological inhibition of tyrosine kinase activity enhances AXL expression, phosphorylation of the PKC−substrate cyclic AMP response element binding (CREB) protein, and displays synergy with AXL and PKC inhibitors. Collectively, our data suggest an involvement of AXL in tyrosine kinase inhibitor resistance and link PKC activation as a possible signaling mediator.

Therapy resistance remains one of the major challenges for cancer treatment that largely limits treatment benefits and patient survival. The underlying mechanisms that lead to therapy resistance are highly complicated because of the specificity to the cancer subtype and therapy. The expression of the anti-apoptotic protein BCL2 has been shown to be deregulated in T-cell acute lymphoblastic leukemia (T-ALL), where different T-ALL cells display a differential response to the BCL2-specific inhibitor venetoclax. In this study, we observed that the expression of anti-apoptotic BCL2 family genes, such as BCL2, BCL2L1, and MCL1, is highly varied in T-ALL patients, and inhibitors targeting proteins coded by these genes display differential responses in T-ALL cell lines. Three T-ALL cell lines (ALL-SIL, MOLT-16, and LOUCY) were highly sensitive to BCL2 inhibition within a panel of cell lines tested. These cell lines displayed differential BCL2 and BCL2L1 expression. Prolonged exposure to venetoclax led to the development of resistance to it in all three sensitive cell lines. To understand how cells developed venetoclax resistance, we monitored the expression of BCL2, BCL2L1, and MCL1 over the treatment period and compared gene expression between resistant cells and parental sensitive cells. We observed a different trend of regulation in terms of BCL2 family gene expression and global gene expression profile including genes reported to be expressed in cancer stem cells. Gene set enrichment analysis (GSEA) showed enrichment of cytokine signaling in all three cell lines which was supported by the phospho-kinase array where STAT5 phosphorylation was found to be elevated in resistant cells. Collectively, our data suggest that venetoclax resistance can be mediated through the enrichment of distinct gene signatures and cytokine signaling pathways.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer with a poor prognosis. Approximately 30% of AML patients carry mutations in the type III receptor tyrosine kinase FLT3. The most common form of FLT3 mutations includes internal tandem duplication (ITD) mutations, which are also associated with poor clinical outcomes. Several inhibitors of FLT3 have been developed and are under clinical trials. Major problems in targeting FLT3-ITD include acquired resistance to the inhibitors which is mediated by secondary point mutations in FLT3. The activity of many, but not all, tyrosine kinases is dependent on the well-conserved tyrosine residue in the activation loop for receptor activation. For instance, we have recently shown that the KIT receptor is dependent on its activation loop tyrosine for downstream signaling and receptor stability, but not for the activation of the receptor. In many AML patients, FLT3-ITD mutations in combination with mutations in the activation loop tyrosine residue (Y842) result in drug resistance. We therefore stably transduced cells with FLT3-ITD in combination with different Y842 mutants (Y842C and Y842F). Using MM-PBSA, we found that the introduction of the Y842C and Y842F point mutations changed the binding energy of the FLT3 inhibitors quizartinib, sorafenib, and midostaurin in silico. In addition, the mutants displayed a change in drug response and apoptosis levels after treatments with FLT3 inhibitors in vitro. Previous data from our lab also shows that the Y842F mutant resulted in delayed tumor formation in vivo, indicating that the phosphorylation of the activation loop tyrosine is an important event in FLT3-ITD-mediated transformation. All in all, our data suggest that the activation loop tyrosine residue in FLT3-ITD positive cells plays an important role in drug response and tumor formation. Citation Format: Lina Al Ashiri, Sausan Moharram, Rituraj Purohit, Julhash Kazi Uddin, Lars Rönnstrand. Y842 mutations in the activation loop of FLT3 regulates drug response in FLT3-ITD positive acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3969.