The nuclease hypersensitivity element III1 upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chairform G-quadruplex, which is destabilized when mutated with a single G 3 A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.

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We have demonstrated that a parallel G-quadruplex structure in the c-MYC promoter functions as a transcriptional repressor element. Furthermore, a specific G-to-A mutation in this element results in destabilization of the G-quadruplex repressor element and an increase in basal transcriptional activity. To validate this model in an in vivo context, we have examined the sequence of this region in human colorectal tumors and the surrounding normal tissue. We have found that ≈30% of tumors contain one of two specific G-to-A mutations, not present in the surrounding normal tissue, that destabilize the parallel G-quadruplex, which would be expected to give rise to abnormally high expression of c-MYC in these cells. In contrast, G-quadruplex-disruptive mutations were absent in 20 colon adenomas, suggesting that these mutations occur late in tumorigenesis. We have also demonstrated that these same mutations are found in established colorectal cell lines. NM23-H2 levels are lower in cancer tissues and cell lines that harbor these mutations. In cells with repressed levels of NM23-H2, the mutated and destabilized G-quadruplex silencer element can be reinstated by the addition of G-quadruplex-stabilizing compounds, providing an opportunity for therapeutic intervention for patients carrying these mutations

The receptor tyrosine kinase AXL is a member of the TAM (Tyro3, AXL, and proto-oncogene tyrosine-protein kinase Mer) family and plays pleiotropic roles in cancer progression. AXL is expressed in immunosuppressive cells, which contributes to decreased efficacy of immunotherapy. Therefore, we hypothesized that AXL inhibition could serve as a strategy to overcome resistance to chimeric antigen receptor T (CART)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CART (CART19)-cell functions. Our results demonstrate that T cells and CART cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CART cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CART cells, Th2 cytokines, reversal of CART-cell inhibition, and promotion of CART-cell effector functions. AXL inhibition is a novel strategy to enhance CART-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CART-cell inhibition through selective targeting of M2-polarized macrophages.

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