AXL Inhibition Improves the Antitumor Activity of Chimeric Antigen Receptor T Cells.

The receptor tyrosine kinase AXL is a member of the 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 (CAR T)-cell therapy. To test this, we determined the impact of AXL inhibition on CD19-targeted CAR T (CART19)-cell functions. Our results demonstrate that T cells and CAR T cells express high levels of AXL. Specifically, higher levels of AXL on activated Th2 CAR T cells and M2-polarized macrophages were observed. AXL inhibition with small molecules or via genetic disruption in T cells demonstrated selective inhibition of Th2 CAR T cells, reduction of Th2 cytokines, reversal of CAR T-cell inhibition, and promotion of CAR T-cell effector functions. AXL inhibition is a novel strategy to enhance CAR T-cell functions through two independent, but complementary, mechanisms: targeting Th2 cells and reversing myeloid-induced CAR T-cell inhibition through selective targeting of M2-polarized macrophages.

The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo.

Cationic porphyrins are being studied as possible anticancer agents because of their ability to bind to and stabilize DNA guanine quadruplexes (G-quadruplexes). We have shown previously that the cationic porphyrin TMPyP4 is able to bind to and stabilize G-quadruplexes in human telomere sequences, resulting in inhibition of telomerase activity. To better understand the mechanism of action behind telomerase inhibition by TMPyP4, we performed a cDNA microarray analysis on cells treated with TMPyP4 and TMPyP2, a positional isomer of TMPyP4 that has low affinity for G-quadruplexes. Analysis of time course data from the microarray experiments revealed that TMPyP4 and TMPyP2 treatment altered the expression of several gene clusters. We found that c-MYC, an oncogene nearly ubiquitous in human tumors that bears the potential in its promoter to form a G-quadruplex, was among the genes specifically down-regulated by TMPyP4, but not by TMPyP2. The hTERT gene, which encodes the catalytic subunit of telomerase, is transcriptionally regulated by c-MYC, and we have found that TMPyP4 also causes a decrease in human telomerase reverse transcriptase transcripts, suggesting two possible mechanisms for the effect of TMPyP4 on telomerase activity. We also show that TMPyP4, but not TMPyP2, is able to prolong survival and decrease tumor growth rates in two xenograft tumor models. We believe that, because of the actions of TMPyP4 in decreasing both c-MYC protein levels and telomerase activity, as well as its anticancer effects in vivo, it is a worthwhile agent to pursue and develop further.

Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription.

The nuclease hypersensitivity element III(1) 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 chair-form G-quadruplex, which is destabilized when mutated with a single G --> 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.

Mutations in the G-quadruplex silencer element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue.

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 approximately 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.