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Supplementary Figure Legends and Figures

PDF file 1227K, This file contains Supplemental Figures 1-7, which provide additional supporting information for each of the main figures

PDOTS Movie

PDOTS Movie 2

In vitro kinase inhibitory activity of Compound 1

PDF file 146K, This file contains the legends to Supplementary Figures 1-7 and Supplementary methods

S1 XLSX file 63K, This Supplementary Table contains the results of ORF screen to identify genes that can rescue the suppression of TBK1 in a KRAS-dependent cell line

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Supplemental Figure 1. Characterization of DFCI 359 and DFCI 315.

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ϵ (IKKϵ) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKϵ substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKϵ activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNβ. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKϵ in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic.Significance:These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.

SF1: Unaltered immunoblots for Fig 4E. SF2: Unaltered immunoblots for Fig 4F. SF3: Unaltered immunoblots for Fig 4G. SF4: Additional phosphopeptide analysis for cell based and in vitro SILAC screen. SF5: Lower magnification (40X) of basal expression of V5 and Rab7 in mutants, to correspond with Fig 4A. SF6: EGFR and STING localization following TBK1/IKKE inhibition. SF7: Schematic for Rab7S72 role in regulating STING/TBK1/IKKE signaling in TNBCs. STableS3: Primer sequence for qRT-PCR. STableS4: List of antibodies. STableS5: PTEN/STING Staining of a TNBC TMA.

Supplementary Table S1

Phosphopeptides from cell based screen

Purpose:Evaluating drug responses using primary patient-derived cells ex vivo represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib combinations in HER2 mutant non–small cell lung cancer (NSCLC) patient xenograft-derived organotypic spheroids (XDOTS) using a short-term ex vivo system.Experimental Design:We generated two HER2-mutant NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREdelinsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated ex vivo with neratinib-based drug combinations. Live/dead quantification was performed by dual-labeling deconvolution fluorescence microscopy. The most efficacious ex vivo combination was subsequently validated in vivo using the DFCI359 and DFCI315 PDXs and a HER2YVMA genetically engineered mouse model.Results:Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab in vivo was more effective compared with single-agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling.Conclusions:The XDOTS platform can be used to evaluate therapies and therapeutic combinations ex vivo using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.

Supplemental Legend

With more effective screening and treatment strategies, there is debate over whether surgical axillary staging should be deescalated for patients with small favorable breast cancers, such as tubular carcinoma (TC). We identified patients with TC [defined as > 90% tubular tubules (angulated, not multilayered)] and known surgical axillary staging from our institutional database (2000–2018). Using the National Cancer Database (NCDB) (2004–2015), we identified patients with TC, ductal carcinoma in situ (DCIS), and pT1 estrogen receptor (ER)-positive invasive ductal carcinoma (IDC). We determined the rates of lymph node (LN) metastases, and the 5- and 10-year overall survival (OS) for patients with LN-negative versus LN-positive disease using the Kaplan–Meier method and propensity match analysis. In our institutional cohort, we identified 112 patients with T1 TC; only one (0.9%) patient had nodal involvement. In the NCDB cohort, we identified 6938 patients with T1 TC; 323 (4.7%) patients had axillary LN disease. The rate of axillary LN involvement for TC was comparable to that identified for patients with DCIS (4.2%), and much lower than that found for patients with grade I–III, T1, ER-positive IDC (20.5%), and patients with grade I, T1, ER-positive IDC (14.4%). There was no difference in 5-year (94.6% versus 95.4%, p = 0.67) and 10-year (83.9% versus 85.2%, p = 0.98) OS between TC patients with or without LN involvement. Kaplan–Meier survival curves even after propensity score matching suggest that tubular histology is independently associated with improved survival. T1 TC is an excellent starting point for deescalation of surgical axillary staging.

Purpose: Evaluating drug responses using primary patient-derived cells ex vivo represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib combinations in HER2 mutant non–small cell lung cancer (NSCLC) patient xenograft-derived organotypic spheroids (XDOTS) using a short-term ex vivo system. Experimental Design: We generated two HER2-mutant NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREdelinsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated ex vivo with neratinib-based drug combinations. Live/dead quantification was performed by dual-labeling deconvolution fluorescence microscopy. The most efficacious ex vivo combination was subsequently validated in vivo using the DFCI359 and DFCI315 PDXs and a HER2YVMA genetically engineered mouse model. Results: Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab in vivo was more effective compared with single-agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling. Conclusions: The XDOTS platform can be used to evaluate therapies and therapeutic combinations ex vivo using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ϵ (IKKϵ) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKϵ substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKϵ activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNβ. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKϵ in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. Significance: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.

With more effective screening and treatment strategies, there is debate over whether surgical axillary staging should be deescalated for patients with small favorable breast cancers, such as tubular carcinoma (TC).We identified patients with TC [defined as 90% tubular tubules (angulated, not multilayered)] and known surgical axillary staging from our institutional database (2000-2018). Using the National Cancer Database (NCDB) (2004-2015), we identified patients with TC, ductal carcinoma in situ (DCIS), and pT1 estrogen receptor (ER)-positive invasive ductal carcinoma (IDC). We determined the rates of lymph node (LN) metastases, and the 5- and 10-year overall survival (OS) for patients with LN-negative versus LN-positive disease using the Kaplan-Meier method and propensity match analysis.In our institutional cohort, we identified 112 patients with T1 TC; only one (0.9%) patient had nodal involvement. In the NCDB cohort, we identified 6938 patients with T1 TC; 323 (4.7%) patients had axillary LN disease. The rate of axillary LN involvement for TC was comparable to that identified for patients with DCIS (4.2%), and much lower than that found for patients with grade I-III, T1, ER-positive IDC (20.5%), and patients with grade I, T1, ER-positive IDC (14.4%). There was no difference in 5-year (94.6% versus 95.4%, p = 0.67) and 10-year (83.9% versus 85.2%, p = 0.98) OS between TC patients with or without LN involvement. Kaplan-Meier survival curves even after propensity score matching suggest that tubular histology is independently associated with improved survival.T1 TC is an excellent starting point for deescalation of surgical axillary staging.

The after mapping of the axilla: radiotherapy or surgery (AMAROS) trial concluded that for patients with cT1-2 N0 breast cancer and one or two positive sentinel lymph nodes (SLNs), axillary radiotherapy (AxRT) provides equivalent locoregional control and a lower incidence of lymphedema compared with axillary lymph node dissection (ALND). The study prospectively assessed how often ALND could be replaced by AxRT in a consecutive cohort of patients undergoing mastectomy for cT1-2 N0 breast cancer. In November 2015, our multidisciplinary group agreed to omit routine intraoperative SLN evaluation for cT1-2 N0 patients undergoing upfront mastectomy and potentially eligible for postmastectomy radiation therapy (PMRT), including those 60 years of age or younger and those older than 60 years with high-risk features. Patients with one or two positive SLNs on final pathology were reviewed to determine whether PMRT including the full axilla was an appropriate alternative to ALND. From November 2015 to December 2016, 154 patients met the study criteria, and 114 (74%) formed the final study cohort. Intraoperative SLN evaluation was omitted for 76 patients (67%). Of these patients, 20 (26%) had one or two positive SLNs, and 14 of these patients received PMRT + AxRT as an alternative to ALND. Three patients returned for ALND, and three patients were observed. On univariate analysis, tumor size, LVI, number of positive lymph nodes, and receipt of chemotherapy were associated with receipt of PMRT. For the majority of patients with one or two positive SLNs, ALND was avoided in favor of PMRT + AxRT. With appropriate multidisciplinary strategies, intraoperative evaluation of the SLN and immediate ALND can be avoided for patients meeting the AMAROS criteria and eligible for PMRT.

Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKϵ inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo. Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196–215. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Deng et al., p. 216. This article is highlighted in the In This Issue feature, p. 127

PURPOSE: Evaluating drug responses using primary patient derived cells ex vivo represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib combinations in HER2 mutant non-small cell lung cancer (NSCLC) patient xenograft derived organotypic spheroids (XDOTS) using a short term ex vivo system. METHODS: We generated two HER2 mutant NSCLC PDX models (DFCI359 (HER2 exon19 755_757LREdelinsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)) and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated ex vivo with neratinib based drug combinations. Live/dead quantification was performed by dual labeling deconvolution fluorescence microscopy. The most efficacious ex vivo combination was subsequently validated in vivo using the DFCI359 and DFCI315 PDXs and a HER2(YVMA) genetically engineered mouse model (GEMM). RESULTS: Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab, and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab in vivo was more effective compared to single agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling. CONCLUSIONS: The XDOTS platform can be used to evaluate therapies and therapeutic combinations ex vivo using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

In postmenopausal women with hormone receptor (HR)-positive breast cancer, neoadjuvant endocrine therapy (ET) provides effective downstaging of tumor for improved surgical outcome and offers an important advantage of assessing tumor endocrine responsiveness for individualized therapy in the adjuvant setting. Although approximately 60 % of breast cancers in premenopausal women are HR positive, the role of neoadjuvant ET in this population is not well defined.We identified 162 patients with stage I-III estrogen receptor-positive breast cancer treated with neoadjuvant ET between 2003 and 2012. Of this group, 17 patients were premenopausal. Data included patient/tumor characteristics, surgical, systemic, and radiation treatment received, and outcomes. Descriptive statistics were used for data summary.The cohort included 17 patients with a mean age of 46.2 years (range 39-53 years). Patients were treated with a combination of gonadotrophic-releasing hormone agonist with either an aromatase inhibitor (n = 14) or tamoxifen (n = 3) for 4-6 months. Among the premenopausal patients, six underwent breast-conserving therapy, with 3 of 6 (50.0 %) having positive margins. Adjuvant chemotherapy was recommended for 13 (76.5 %), and adjuvant radiotherapy was recommended for 13 (76.5 %). Of the 17 premenopausal women, 11 had a clinical response based on response evaluation criteria in solid tumors (RECIST) of a decrease in tumor size of 30 % (64.7 %); this is similar to that of postmenopausal women, where 85 of 145 (58.6 %) patients showed a clinical response.As with all neoadjuvant systemic interventions, we identified those with disease that did and did not respond to ET, emphasizing the heterogeneity of HR-positive breast cancers. The response rate of premenopausal women to neoadjuvant ET is similar to that of postmenopausal women.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of cancers that are defined by the absence of hormone receptor expression and HER2 amplification. Here, we found that inducible IκB kinase–related (IKK-related) kinase IKBKE expression and JAK/STAT pathway activation compose a cytokine signaling network in the immune-activated subset of TNBC. We found that treatment of cultured IKBKE-driven breast cancer cells with CYT387, a potent inhibitor of TBK1/IKBKE and JAK signaling, impairs proliferation, while inhibition of JAK alone does not. CYT387 treatment inhibited activation of both NF-κB and STAT and disrupted expression of the protumorigenic cytokines CCL5 and IL-6 in these IKBKE-driven breast cancer cells. Moreover, in 3D culture models, the addition of CCL5 and IL-6 to the media not only promoted tumor spheroid dispersal but also stimulated proliferation and migration of endothelial cells. Interruption of cytokine signaling by CYT387 in vivo impaired the growth of an IKBKE-driven TNBC cell line and patient-derived xenografts (PDXs). A combination of CYT387 therapy with a MEK inhibitor was particularly effective, abrogating tumor growth and angiogenesis in an aggressive PDX model of TNBC. Together, these findings reveal that IKBKE-associated cytokine signaling promotes tumorigenicity of immune-driven TNBC and identify a potential therapeutic strategy using clinically available compounds.

Background: Epithelial cell adhesion molecule (EpCAM) is frequently expressed in breast cancer, and its expression has been associated with poor prognosis. Breast cancer can be subdivided into intrinsic subtypes, differing in prognosis and response to therapy. Methods: To investigate the association between EpCAM expression and prognosis in the intrinsic subtypes of breast cancer, we performed immunohistochemical studies on a tissue microarray encompassing a total of 1365 breast cancers with detailed clinicopathological annotation and outcomes data. Results: We observed EpCAM expression in 660 out of 1365 (48%) cases. EpCAM expression varied significantly in the different intrinsic subtypes. In univariate analyses of all cases, EpCAM expression was associated with a significantly worse overall survival. In the intrinsic subtypes, EpCAM expression was associated with an unfavourable prognosis in the basal-like and luminal B HER2+ subtypes but associated with a favourable prognosis in the HER2 subtype. Consistently, specific ablation of EpCAM resulted in increased cell viability in the breast cancer cell line SKBR3 (ER−, PR−, and HER2+) but decreased viability in the breast cancer cell line MDA-MB-231 (ER−, PR−, and HER2− ). Conclusion: The differential association of EpCAM expression with prognosis in intrinsic subtypes has important implications for the development of EpCAM-targeted therapies in breast cancer.

584Background: Recent trials have demonstrated the feasibility of SLN biopsy in cN1 patients who become cN0 after NAC. We sought to evaluate success of SLN mapping and accuracy of intraop frozen se...

In addition to causing regression of the Mullerian duct in the male embryo, Mullerian Inhibiting Substance (MIS) inhibits the growth of epithelial ovarian cancer cells, which are known to be of Mullerian origin. Because the uterine cervix is derived from the same Mullerian duct precursor as the epithelium of the ovary, we tested the hypothesis that cervical cancer cells might also respond to MIS. A number of cervical cancer cell lines express the MIS type II receptor, and MIS inhibits the growth of both human papilloma virus-transformed and non-human papilloma virus-transformed cervical cell lines, with a more dramatic effect seen in the latter. As in the ovarian cancer cell line OVCAR8, suppression of growth of the C33A cervical cancer cell line by MIS is associated with induction of the p16 tumor suppressor protein. However, in contrast to OVCAR8 cells, induction of p130 and p107 appears to play an important role in the inhibition of growth of C33A cells by MIS. Finally, normal cervical tissue expresses the MIS type II receptor in vivo , supporting the idea that MIS could be a targeted therapy for cervical cancer.

Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)–related kinases Tank-binding kinase-1 (TBK1) and IKKϵ promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKϵ inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKϵ promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKϵ, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS. Significance: In addition to activating MAPK and PI3K, oncogenic KRAS engages cytokine signaling to promote tumorigenesis. CYT387, originally described as a selective JAK inhibitor, is also a potent TBK/IKKϵ inhibitor that uniquely disrupts a cytokine circuit involving CCL5, IL-6, and STAT3. The efficacy of CYT387-based treatment in murine Kras-driven lung cancer models uncovers a novel therapeutic approach for these refractory tumors with immediate translational implications. Cancer Discov; 4(4); 452–65. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377