TALL1 cells are NOTCH3-dependent.

<p>(A) NOTCH1 and NOTCH3 mRNA transcript levels. Transcripts were quantified using gene specific primer sets and GAPDH as a reference gene. (B) Active nuclear ICN1 and ICN3. Western blots of fractionated cell lysates were stained with the indicated specific antibodies. The anti-N3-S3 antibody, which recognizes gamma-secretase cleaved NOTCH3, has weak cross-reactivity to gamma-secretase cleaved NOTCH1 (asterisk). (C) TALL1 cell growth is strongly inhibited by GSI, partially inhibited by the anti-NOTCH3 NRR antibody A4, and resistant to the anti-NOTCH1 NRR antibody WC75.</p

Interchangeability of ICN1 and ICN3 in TALL1 cells and CUTLL1 cells.

<p>Cell growth plotted as a function of time under different treatment conditions, showing rescue of GSI treated TALL1 and CUTLL1 cells by either ICN1 or ICN3.</p

Comparison of the effect of GSI on <i>MYC</i> expression and cell growth in TALL1 cells and CUTLL1 cells.

<p>(A) qPCR analysis of <i>MYC</i> mRNA, normalized relative to 18s rRNA, in CUTLL1 cells and TALL1 cells. Cells were treated with DMSO or GSI for 3 days and incubated for 24 hours after GSI washout. (B) TALL1 is more sensitive to Notch inhibition than CUTLL1 cells. Cell viability, assessed using cell titer blue, was measured as a function of the dose of compound E.</p

NOTCH1 and NOTCH3 peaks are highly overlapping.

<p>(A) Scatter plot showing read count, of total (n = 32788; top) and dynamic (n = 2332; bottom) NOTCH1 and NOTCH3 peaks. The average read count under each peak is indicated in color on a sliding scale from blue (low) to red (high). (B) Whisker plots showing signal strength at NOTCH1 and NOTCH3 shared and selective peaks. Peaks responding to both NOTCH1 and NOTCH3 exhibit much stronger signal strength than NOTCH1 or NOTCH3 selective peaks. (C) Z-scores showing enrichment for the RBPJ motif both at NOTCH1 and NOTCH3 shared peaks and at NOTCH1 or NOTCH3 selective peaks.</p

Notch binding to TALL1 and CUTLL1 genomes.

<p>(A) Genomic distribution of all NOTCH3 peaks (n = 20577) and dynamic NOTCH3 peaks (n = 992) in TALL1 cells. The graphs show each peak as a function of its distance from the nearest annotated TSS by the total ChIP-seq intensity under the peak. The red line represents 2kb from a TSS, which is the cutoff used for assigning a peak to a promoter or an enhancer. (B) Genomic distribution of all NOTCH1 peaks (n = 17315) and dynamic NOTCH1 peaks (n = 1650) in CUTLL1 cells, plotted in a similar fashion to (A). (C) Across all dynamic NOTCH3 (left) and NOTCH1 (right) peaks, there is a decrease in the normalized read density of H3K27 acetylation in the Notch-off state. The valley in both graphs is centered over the summit (0 bp) of the corresponding NOTCH1 and NOTCH3 peaks.</p

Overlap of NOTCH1- and NOTCH3-regulated genes.

<p>(A) Comparison of Notch responsive changes in gene expression between CUTLL1 and TALL1 cell lines. Genes shown are in the top quintile of gene expression, averaged across all samples and represented on a sliding color scale from yellow (low expression) to blue (highest expression). Known NOTCH1 target genes (as indicated) are dynamically regulated in both CUTLL1 and TALL1 cells. (B) Heat map showing genes that exhibit significant changes (p<0.05) in expression between Notch-on and Notch-off states. Genes represented in the heat map are the union of all Notch-dependent genes in both cell lines (n = 1291). The heatmap is ordered vertically by the average fold change in expression across all five rows. (C) Whisker plots comparing average expression and fold-change of genes responding to both NOTCH1 and NOTCH3 (common genes) or selectively to either NOTCH1 or NOTCH3 (selective genes). Though common genes and NOTCH1- and NOTCH3-selective genes have similar overall levels of expression, common genes have a significantly larger fold change in response to GSI treatment. P values were calculated by t-test.</p

Structural and Functional Consequences of Three Cancer-Associated Mutations of the Oncogenic Phosphatase SHP2

The proto-oncogene <i>PTPN11</i> encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the <i>PTPN11</i> variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small-angle X-ray scattering, and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies

Dual Allosteric Inhibition of SHP2 Phosphatase

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the <i>PTPN11</i> gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (<b>1</b>) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (<b>2</b>) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that <b>2</b> binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding sitea cleft formed at the interface of the <i>N</i>-terminal SH2 and PTP domains. Derivatization of <b>2</b> using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by <b>1</b> and <b>2</b> was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies