Melanoma, PD-1, and FTO - A layman’s perspective In anticipation...

Melanoma, PD-1, and FTO - A layman’s perspective

In anticipation for the preclinical FTO programs that are rapidly approaching, I have decided to break down the two important articles that relate and reinforce RACs direction moving forward. This is to put the science in terms that you can understand and improve your understanding of what we as shareholders and ultimately what a large pharmaceutical company would like to see from preclinical data. I have selected data that I think is critical to understanding what RAC will need to prove in preclinical studies as well as some extra bits and will try to keep the information as brief and clear as possible.

Note: Apologies for spacing and structure. Who knows how this will turn out when I hit post.

FTO as a protumorigenic (promoting tumor growth) factor in melanoma

FTO expression was similar in different stages of melanoma (stages I-IV) and was upregulated in six of the melanoma cell lines (indicated by the thicker darker lines in the FTO row) compared to NHEM#1 and NHEM#2 (normal melanocytes; not cancerous). Mel624 and B16F10 are particularly aggressive melanoma cell lines, which showed much greater concentration of FTO (indicated by the much thicker and darker lines).

Knockdown of FTO in FTO-high Mel624, CHL-1, and B16F10 cells decreased cell growth/ proliferation, migration, invasion, and cell viability in cells placed in suspension. Highlighting the role of FTO in each of these functions. In the following graphs the sh before FTO-1 and FTO-2 indicates that FTO has been knocked down.

In vitro (in a test tube / dish): You can see that in cell lines with shFTO-1 and shFTO-2 (green and red), there is reduced cell number, migration, invasion, and cell viability of melanoma cells.

In vivo (living thing): You can see that in cell lines with shFTO-1 and shFTO-2 (red and green), there is reduced tumor volume and tumor weight of melanoma cells.

Remember that Mel624 and B16F10 are particularly aggressive melanomas with greatly increased FTO expression.

Identification of functionally critical target genes of FTO

Following gene targeting testing, PD-1, CXCR4, and SOX10 were identified. PD-1 has been shown to be expressed in melanoma cells and act as an intrinsic protumorigenic (promote tumor growth) factor to promote melanoma. Also, CXCR4 and SOX10 are critical melanoma-promoting genes. FTO was found to regulate the expression of PD-1 (PDCD1), CXCR4, and SOX10, where FTO knockdown reduced the protein levels of PDCD1, CXCR4, and SOX10.

Next the study discovered that PDCD1, CXCR4, and SOX10 are critical downstream targets (expressed as a result of FTO overexpression) of FTO that are responsible for its function in melanoma. Very simplistically, think of FTO as the general and PDCD1, CXCR4, and SOX10 as the soldiers.

To draw a conclusion from these graphs you need to look at the red line compared to the green line. These lines indicate the cells that have had their FTO knocked down. The green line indicates cells that have had either PDCD1, CXCR4, or SOX10 upregulated (which can be seen in the highlighter), which as you can see by the graph by comparing it to the red line (vector or control cell) has led to an increase in cell number. Also, next to each graph you can see that migration increases in the light grey boxes (which include the cells with either PDCD1, CXCR4, or SOX10 upregulated). This confirms PDCD1, CXCR4, or SOX10 as downstream targets of FTO.

Role of FTO on anti-PD-1 blockade and IFNγ

Because of the role of FTO in promoting melanoma tumorigenicity and regulating the expression of tumor-promoting melanoma cell-intrinsic PD-1 as well as CXCR4 and SOX10, the authors reasoned that FTO may also regulate the response of melanoma to immunotherapy.

Treating mice bearing B16F10 melanomas with anti-PD-1 antibody had no effect on tumor growth of control B16F10 cells mimicking resistance to PD-1 blockade in melanoma immunotherapy. However, anti-PD-1 antibody significantly inhibited tumor growth for FTO knockdown tumors. The C57BL/6 mice are immunocompetent (meaning they have a normal immune response). The graph below shows that knockdown of FTO in these mice reduces tumor volume (thick blue line), and a combination of knockdown of FTO and anti-PD-1 antibody showed significantly lower tumor volume than FTO knockdown alone (dotted blue line).

Next, FTO knockdown sensitized melanoma cells to IFN-g (interferon gamma) induced growth inhibition and cell killing. IFN-g is an important cytokine that’s involved in inducing and modulating immune responses, which has been shown to be upregulated in PD-1 and PD-L1 therapies. Looking at the graph below you can see that FTO down regulation increased the cell killing properties of IFN-g (compare shNC + IFN-g to shFTO + IFN-g).

Below you can see by artificially increasing the downstream targets of FTO (PDCD1, CXCR4, SOX10), the cell-killing effect of IFN-g in FTO knockdown melanoma cells is reduced. What the below graph is showing you is that as the downstream targets are increased, the effect of IFN-g is reduced, which highlights the therapeutic opportunity of inhibiting FTO.

Lastly, the researchers summarise the relationship in a figure and I have shown it below. Briefly, FTO influences the m6A concentrations in the cell, which changes the expression and concentration of downstream targets PD-1, CXCR4, and SOX10, which influences proliferation, migration, growth, and anti-PD-1 response. Therefore, pharmacological inhibition of FTO and regulation of intracellular m6A is an extremely important therapeutic target for influencing melanoma cancer. Importantly, this mechanism has been shown in test tubes as well as living things. Confirming (and possibly improving on) the results and mechanisms in this study will open the door for further anti-PD-1 combination studies and add extreme value to RAC board and shareholders as well opportunities and hope for melanoma sufferers around the world.

All IMO - DYOR