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This file contains the Supplementary Materials and Methods.

Figure S1: Hit validation of siRNA screen for determinants of WEE1 inhibitor sensitivity. Figure S2: WEE1 inhibition by MK-1775 induces replication stress without DSBs and is dependent on CDK1/2 activity. Figure S3: Dual inhibition of WEE1 and MYT1 by PD0166285 induces premature mitosis. Figure S4: Increased sensitivity to WEE1 inhibition in FANCM- or BRIP1-depleted cells is caused by replication stress and unscheduled mitosis. Figure S5: Nucleoside supplementation defers the onset of replication stress and unscheduled mitosis triggered by WEE1 inhibition in cells with FA defects. Figure S6: Co-depletion of MRE11, but not MUS81 or SLX4, partially rescued the replication stress and unscheduled mitosis induced by WEE1 inhibition in FANCM- and BRIP1-depleted cells.

This file contains the legends for Supplementary Figures S1-S6.

WEE1 kinase regulates CDK1 and CDK2 activity to facilitate DNA replication during S-phase and to prevent unscheduled entry into mitosis. WEE1 inhibitors synergize with DNA-damaging agents that arrest cells in S-phase by triggering direct mitotic entry without completing DNA synthesis, resulting in catastrophic chromosome fragmentation and apoptosis. Here, we investigated how WEE1 inhibition could be best exploited for cancer therapy by performing a functional genetic screen to identify novel determinants of sensitivity to WEE1 inhibition. Inhibition of kinases that regulate CDK activity, CHK1 and MYT1, synergized with WEE1 inhibition through both increased replication stress and forced mitotic entry of S-phase cells. Loss of multiple components of the Fanconi anemia (FA) and homologous recombination (HR) pathways, in particular DNA helicases, sensitized to WEE1 inhibition. Silencing of FA/HR genes resulted in excessive replication stress and nucleotide depletion following WEE1 inhibition, which ultimately led to increased unscheduled mitotic entry. Our results suggest that cancers with defects in FA and HR pathways may be targeted by WEE1 inhibition, providing a basis for a novel synthetic lethal strategy for cancers harboring FA/HR defects. Mol Cancer Ther; 14(4); 865–76. ©2015 AACR.

CDK4/6 inhibition partly overcomes NF1 loss in T47D cells

Supplementary Table 1. Targets of The Breast NGS v1.0 capture panel. Supplementary Table 2. Gene list of NanoString nCounter{trade mark, serif} custom codeset. Supplementary Table 3. Gene list of Human Estrogen Receptor RT2 Profiler PCR Array (330231, PAHS-005ZA-24, Qiagen) Supplementary Table 4. Lines of treatment of patients with ESR1 and NF1 mutations. Supplementary Table 5. Disease free survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype. Supplementary Table 6. Advanced overall survival for mutated genes with incidence {greater than or equal to}5% presented by HR+/HER2-, HER2+ and TNBC subtype

Purpose: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer.Experimental Design: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity.Results: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K–AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo.Conclusions: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers. Clin Cancer Res; 17(16); 5275–86. ©2011 AACR.

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Genetic profile of advanced breast cancer subtypes

NCOR expression following NF1 loss

Purpose:Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Experimental Design:Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.Results:We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations in HER2 (in 6.19% of samples), AKT1 (7.14%), and NF1 (8.10%). Of these enriched mutations, we show that NF1 mutations were frequently acquired in ABC, not present in the original primary disease. In ER-positive cancer cell line models, loss of NF1 resulted in endocrine therapy resistance, through both ER-dependent and -independent mechanisms. NF1 loss promoted ER-independent cyclin D1 expression, which could be therapeutically targeted with CDK4/6 inhibitors in vitro. Patients with NF1 mutations detected in baseline circulating tumor DNA had a good outcome on the CDK4/6 inhibitor palbociclib and fulvestrant.Conclusions:Our research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of NF1 mutations.

NCOR expression in NF1 mutated cancers

Genetic profile of ER positive primary breast cancers that 'switch' to triple negative recurrent advanced breast cancer

Advanced breast cancer (ABC) has not been subjected to the same degree of molecular scrutiny as early primary cancer. Breast cancer evolves with time and under the selective pressure of treatment, with the potential to acquire mutations with resistance to treatment and disease progression. To identify potentially targetable mutations in advanced breast cancer, we performed prospective molecular characterization of a cohort of patients with ABC.Biopsies from patients with advanced breast cancer were sequenced with a 41 genes targeted panel in the ABC Biopsy (ABC-Bio) study. Blood samples were collected at disease progression for circulating tumor DNA (ctDNA) analysis, along with matched primary tumor to assess for acquisition in ABC in a subset of patients.We sequenced 210 ABC samples, demonstrating enrichment compared with primary disease for potentially targetable mutations inOur research identifies multiple therapeutic opportunities for advanced breast cancer and identifies the previously underappreciated acquisition of