F, unsorted bone marrow (BM) or peripheral blood (PB) mononuclear cells from MM patient samples were treated with SCR or WW peptide (500 nM) for 4 days followed by plating in methylcellulose.The function of scaffolding proteins is to bring together two or more proteins in a relatively stable configuration, hence their name. P-values were determined by Student’s t-testing. The percentage of CD138 neg, PI negative cells was then calculated after flow cytometric analysis (absolute values ranged from 0.5–1%) and normalized to a control sample. E, MM cells were treated with peptide (100 nM) and subjected to staining with CD138-FITC and PI. D, MM cells were treated with 100 nM of MEK inhibitor selumetinib or DMSO control for 4 days followed by quantification of colony formation as in A. C, cells from A were subjected to cell staining using an annexin-V followed by flow cytometric analysis. Colonies were scored and then harvested for re-plating using equal volumes of samples after washing. B, MM cells were treated with peptide and then plated onto methylcellulose as in A. p-values were determined by one-way ANOVA followed by multiple comparison testing. Results are the average of 3 biological replicates. Unselected cells were then plated in methylcellulose in the absence of peptide.
A, the indicated MM cell lines were treated with 500 nM of scrambled (SCR) or WW peptide for 4 days (RPMI-8226, NCI-H929, and MM1.S are RAS mutated whereas KMS.12 is RAS/BRAF wild-type). IQGAP1 WW peptide impacts MM clonogenic growth and self-renewal. Results are the average of 2 biological replicates. E, KMS.12 cells (RAS/BRAF wild-type) containing the indicated doxycycline inducible shRNA were treated with doxycycline and subjected to colony forming cell assays as in A. C and D, the cells from panel A and B were subjected to annexin V staining followed by flow cytometric analysis. B, RPMI-8226 cells containing doxycycline inducible shRNA targeting IQGAP1 and ectopic control or hIQGAP1 rescue were subjected to doxycycline treatment for 9 days followed by colony formation as in A. Quantification of colonies occurred 7–10 days later. Phenotypically unselected cells were then plated in methylcellulose without doxycycline. A, RPMI-8226 cells (RAS mutant) containing doxycycline inducible shRNA targeting scramble (shSCR) or IQGAP1 were treated with doxycycline for 6 days. IQGAP1 is required for clonogenic growth in MM. ©2016 American Association for Cancer Research. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation.
Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease.