IQGAP1 is an oncogenic target in canine melanoma.

Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro

A Balanced Look at the Implications of Genomic (and Other “Omics”) Testing for Disease Diagnosis and Clinical Care

The tremendous increase in DNA sequencing capacity arising from the commercialization of “next generation” instruments has opened the door to innumerable routes of investigation in basic and translational medical science. It enables very large data sets to be gathered, whose interpretation and conversion into useful knowledge is only beginning. A challenge for modern healthcare systems and academic medical centers is to apply these new methods for the diagnosis of disease and the management of patient care without unnecessary delay, but also with appropriate evaluation of the quality of data and interpretation, as well as the clinical value of the insights gained. Most critically, the standards applied for evaluating these new laboratory data and ensuring that the results and their significance are clearly communicated to patients and their caregivers should be at least as rigorous as those applied to other kinds of medical tests. Here, we present an overview of conceptual and practical issues to be considered in planning for the integration of genomic methods or, in principle, any other type of “omics” testing into clinical care

IQGAP1 is an oncogenic target in canine melanoma

<div><p>Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human Braf^V600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.</p></div

Canine melanoma lines are dependent on ERK1/2 similar to human Braf^V600E melanoma lines.

<p>(A) Immunoblot for human Braf^V600E melanoma cells (SK28) and two canine mucosal melanoma cell lines (CMM3, CMM5) treated with DMSO or GSK1120212 (Trametinib) for 8hrs. (B) Growth curves for two canine mucosal melanoma cell lines (CMM3, CMM5) and human Braf^V600E melanoma cells (SK-mel-28) treated with GSK1120212 ranging from 0.0001μM to 30μM. Values represent 3 biological replicates for CMM lines and 2 biological replicates from SK-Mel-28. (C) IC50 values from 4-day proliferation assays in cells as in (A).</p

Canine IQGAP1 is more highly conserved than mouse IQGAP1, particularly within the WW domain.

<p>(A) Schematic of function of WW peptide interruption of binding between IQGAP1 and ERK1/2. (B) IQGAP1 genomic locus with mouse and canine conservation tracks. Specific amino acid alignments of the WW domain [inset]. (C) Diagram of WW peptide mutations. (D) Immunoblot of primary human keratinocytes infected with either the full-length WW domain, one with two mutated central YY residues, a 5’ truncation missing 5AA and a 3’ truncation missing 3AA. (E) Histology and IQGAP1 immunohistochemistry from normal canine oral mucosa and primary canine mucosal melanoma. Scale bar = 100μm.</p