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Purpose: Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (MF) or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of JAK2V617F-mutant cell lines. Patients and Methods: In a phase I dose-escalation and -expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor, umbralisib, in combination with ruxolitinib in patients with MF who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that MTD at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose-escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas. Results: Thirty-seven patients with MF (median age, 67 years) with prior exposure to ruxolitinib were enrolled. A total of 2 patients treated with 800 mg umbralisib experienced reversible grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved a durable complete response, and 12 patients (32%) met the International Working Group-Myeloproliferative Neoplasms Research and Treatment response criteria of clinical improvement. With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up. Conclusions: Adding umbralisib to ruxolitinib in patients was well tolerated and may resensitize patients with MF to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors. Randomized trials testing umbralisib in the treatment of MF should be pursued.

The objective of this study was to determine the survivorship of anatomic total shoulder arthroplasty (aTSA) and reverse TSA (rTSA) over a medium-term follow-up in a large population-based sample and to identify potential risk factors for revision surgery.The State Inpatient Database from the Healthcare Cost and Utilization Project was used to identify patients who underwent aTSA or rTSA from 2011 through 2015 using ICD9 codes. We modeled the primary outcome of time to revision or arthroplasty using the Cox proportional hazards model. The predictors of revision surgery in the model include aTSA versus rTSA, indication for surgery, age, sex, race, urban versus rural residence, hospital length of stay zip code-based income quartile classification, and Elixhauser comorbidity readmission score.Among 43,990 patients in this study, 1,141 (4.0%) underwent revision or implant removal over the 4-year study period. The median age was 71 years, and 57% of patients were female. Indications for the index surgery included primary osteoarthritis (75.2%), cuff tear (8.5%), acute fracture (7.0%), malunion/nonunion (1.4%), and other (7.8%). Among these indications for surgery, the risk of revision or removal was greatest in patients who underwent the primary procedure for malunion/nonunion (hazard ratio [HR] 2.39, 95% confidence interval [CI] 1.69 to 3.39) compared with the reference of primary osteoarthritis. Male patients who underwent aTSA were less likely to need revision surgery than male patients who underwent rTSA (HR: 0.59, 95% CI 0.49 to 0.71), and the opposite relationship was observed in female patients (HR: 1.41, 95% CI 1.18 to 1.69). Age, length of stay, and Elixhauser comorbidity score were predictive of revision surgery (P0.0001, P = 0.0005, P0.0001, respectively), whereas race, urban versus rural, and zip code-based income quartile were not.aTSA and rTSA showed excellent 4-year survivorship of 96.0% in a large population-based sample. aTSA and rTSA survivorships were similar at the 4-year follow-up.

Small bowel adenocarcinomas (SBAs) are rare and frequently treated like large intestinal adenocarcinomas. However, SBAs have a very different microenvironment and could respond differently to the same therapies. Our previous data suggested that SBAs might benefit from targeting the PD-1/PD-L1 axis based on PD-L1 staining in almost 50% of SBA tissue samples tested. Thus, we designed a phase 2 study to explore safety and efficacy of avelumab in SBA.Patients with advanced or metastatic disease were enrolled; ampullary tumors were considered part of the duodenum and allowed. Prior PD-1/PD-L1 inhibition was not allowed. Avelumab (10 mg/kg) was given every 2 weeks, and imaging was performed every 8 weeks. Primary endpoint was response rate.Eight patients (n = 5, small intestine; n = 3, ampullary) were enrolled, with a majority (88%) being male and a median age of 61 years. Of 7 efficacy-evaluable patients, 2 (29%) experienced partial responses; stable disease occurred in 3 additional patients (71%). Median progression-free survival was 3.35 months. Most frequent, related toxicities were anemia, fatigue, and infusion-related reaction (25% each), mostly grade ≤2; grade 3 hypokalemia and hyponatremia occurred in one patient, and another reported grade 4 diabetic ketoacidosis.Despite the observed benefit, accrual was slower than expected and the study was closed early due to feasibility. A general clinic observation was that patients were receiving immunotherapy off-label as the availability of these agents increased. Off-label availability and disease rarity were likely drivers of insufficient accrual.

Supplemental Tables S1-S9 Supplemental Figures S1-S2

Purpose:Treatment options are limited beyond JAK inhibitors for patients with primary myelofibrosis (MF) or secondary MF. Preclinical studies have revealed that PI3Kδ inhibition cooperates with ruxolitinib, a JAK1/2 inhibitor, to reduce proliferation and induce apoptosis of JAK2V617F-mutant cell lines.Patients and Methods:In a phase I dose-escalation and -expansion study, we evaluated the safety and efficacy of a selective PI3Kδ inhibitor, umbralisib, in combination with ruxolitinib in patients with MF who had a suboptimal response or lost response to ruxolitinib. Enrolled subjects were required to be on a stable dose of ruxolitinib for ≥8 weeks and continue that MTD at study enrollment. The recommended dose of umbralisib in combination with ruxolitinib was determined using a modified 3+3 dose-escalation design. Safety, pharmacokinetics, and efficacy outcomes were evaluated, and spleen size was measured with a novel automated digital atlas.Results:Thirty-seven patients with MF (median age, 67 years) with prior exposure to ruxolitinib were enrolled. A total of 2 patients treated with 800 mg umbralisib experienced reversible grade 3 asymptomatic pancreatic enzyme elevation, but no dose-limiting toxicities were seen at lower umbralisib doses. Two patients (5%) achieved a durable complete response, and 12 patients (32%) met the International Working Group-Myeloproliferative Neoplasms Research and Treatment response criteria of clinical improvement. With a median follow-up of 50.3 months for censored patients, overall survival was greater than 70% after 3 years of follow-up.Conclusions:Adding umbralisib to ruxolitinib in patients was well tolerated and may resensitize patients with MF to ruxolitinib without unacceptable rates of adverse events seen with earlier generation PI3Kδ inhibitors. Randomized trials testing umbralisib in the treatment of MF should be pursued.

Supplementary Table 2

Supplementary Figures 1-6

Recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment (TME) contributes to cancer immune evasion. MDSCs express the chemokine receptor CXCR2, and inhibiting CXCR2 suppresses the recruitment of MDSCs into the tumor and the premetastatic niche. Here, we compared the growth and metastasis of melanoma and breast cancer xenografts in mice exhibiting or not exhibiting targeted deletion of Cxcr2 in myeloid cells (CXCR2myeΔ/Δ vs. CXCR2myeWT). Detailed analysis of leukocyte populations in peripheral blood and in tumors from CXCR2myeΔ/Δ mice revealed that loss of CXCR2 signaling in myeloid cells resulted in reduced intratumoral MDSCs and increased intratumoral CXCL11. The increase in intratumoral CXCL11 was derived in part from tumor-infiltrating B1b cells. The reduction in intratumoral MDSCs coupled with an increase in intratumoral B1b cells expressing CXCL11 resulted in enhanced infiltration and activation of effector CD8+ T cells in the TME of CXCR2myeΔ/Δ mice, accompanied by inhibition of tumor growth in CXCR2myeΔ/Δ mice compared with CXCR2myeWT littermates. Treatment of tumor-bearing mice with a CXCR2 antagonist (SX-682) also inhibited tumor growth, reduced intratumoral MDSCs, and increased intratumoral B1b cells expressing CXCL11, leading to an increase in activated CD8+ T cells in the tumor. Depletion of B220+ cells or depletion of CD8+ T cells reversed the tumor-inhibitory properties in CXCR2myeΔ/Δ mice. These data revealed a mechanism by which loss of CXCR2 signaling in myeloid cells modulates antitumor immunity through decreasing MDSCs and enriching CXCL11-producing B1b cells in the TME, which in turn increases CD8+ T-cell recruitment and activation in tumors.

Supplementary Figure Legends

Supplementary Table 1

Mutations assessed at VICC.

Activating NRAS mutations are found in 15% to 20% of melanomas. Immune therapies have become a mainstay in advanced melanoma treatment. We sought to evaluate whether tumor genotype (e.g., NRAS mutations) correlates with benefit from immune therapy in melanoma. We identified 229 patients with melanoma treated with immune therapies [IL2, ipilimumab, or anti-programmed cell death-1/ligand-1 (PD-1/PD-L1)] at three centers and compared clinical outcomes following immune therapy for patients with or without NRAS mutations. Of the 229 patients with melanoma, 60 had NRAS mutation, 53 had BRAF mutation, and 116 had NRAS/BRAF wild type. The NRAS-mutant cohort had superior or a trend to superior outcomes compared with the other cohorts in terms of response to first-line immune therapy (28% vs. 16%, P = 0.04), response to any line of immune therapy (32% vs. 20%, P = 0.07), clinical benefit (response + stable disease lasting ≥24 weeks; 50% vs. 31%, P < 0.01), and progression-free survival (median, 4.1 vs. 2.9 months, P = 0.09). Benefit from anti–PD-1/PD-L1 was particularly marked in the NRAS cohort (clinical benefit rate 73% vs. 35%). In an independent group of patient samples, NRAS-mutant melanoma had higher PD-L1 expression (although not statistically significant) compared with other genotypes (8/12 vs. 9/20 samples with ≥1% expression; 6/12 vs. 6/20 samples with ≥5% expression), suggesting a potential mechanism for the clinical results. This retrospective study suggests that NRAS mutations in advanced melanoma correlate with increased benefit from immune-based therapies compared with other genetic subtypes. If confirmed by prospective studies, this may be explained in part by high rates of PD-L1 expression. Cancer Immunol Res; 3(3); 288–95. ©2015 AACR.

Supplemental Data and Supplemental Figures

Supplementary figures and table

Supplementary Table 1 and Figures 1-7

The chemokine receptor, CXCR4, is involved in cancer growth, invasion, and metastasis. Several promising CXCR4 antagonists have been shown to halt tumor metastasis in preclinical studies, and clinical trials evaluating the effectiveness of these agents in patients with cancer are ongoing. However, the impact of targeting CXCR4 specifically on immune cells is not clear. Here, we demonstrate that genetic deletion of CXCR4 in myeloid cells (CXCR4MyeΔ/Δ) enhances the antitumor immune response, resulting in significantly reduced melanoma tumor growth. Moreover, CXCR4MyeΔ/Δ mice exhibited slowed tumor progression compared with CXCR4WT mice in an inducible melanocyte BrafV600E/Pten−/− mouse model. The percentage of Fas ligand (FasL)–expressing myeloid cells was reduced in CXCR4MyeΔ/Δ mice as compared with myeloid cells from CXCR4WT mice. In contrast, there was an increased percentage of natural killer (NK) cells expressing FasL in tumors growing in CXCR4MyeΔ/Δ mice. NK cells from CXCR4MyeΔ/Δ mice also exhibited increased tumor cell killing capacity in vivo, based on clearance of NK-sensitive Yac-1 cells. NK cell–mediated killing of Yac-1 cells occurred in a FasL-dependent manner, which was partially dependent upon the presence of CXCR4MyeΔ/Δ neutrophils. Furthermore, enhanced NK cell activity in CXCR4MyeΔ/Δ mice was also associated with increased production of IL18 by specific leukocyte subpopulations. These data suggest that CXCR4-mediated signals from myeloid cells suppress NK cell–mediated tumor surveillance and thereby enhance tumor growth. Systemic delivery of a peptide antagonist of CXCR4 to tumor-bearing CXCR4WT mice resulted in enhanced NK-cell activation and reduced tumor growth, supporting potential clinical implications for CXCR4 antagonism in some cancers. Cancer Immunol Res; 6(10); 1186–98. ©2018 AACR.

Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials.Significance:Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494

Supplementary FigureS2: Response to KRT-232.

This file describes additional methodology

[18F]V-1008 localization observed in Ptf1a-Cre;LSL-KrasG12D/+;Tgfbr2+/- (KTC) pancreatic lesions

Purpose: Preclinical studies show that bortezomib, a proteasome inhibitor, blocks NF-κB activation and, combined with temozolomide, enhances activity against human melanoma xenografts and modulates other critical tumor targets. We initiated a phase I trial of temozolomide plus bortezomib in advanced melanoma. Objectives included defining a maximum tolerated dose for the combination, characterizing biomarker changes reflecting inhibition of both proteasome and NF-κB activity in blood (if possible tumor), and characterizing antitumor activity.Experimental Design: Cohorts were enrolled onto escalating dose levels of temozolomide (50-75 mg/m2) daily, orally, for 6 of 9 weeks and bortezomib (0.75-1.5 mg/m2) by i.v. push on days 1, 4, 8, and 11 every 21 days. Peripheral blood mononuclear cells were assayed at specified time points for proteasome inhibition and NF-κB biomarker activity.Results: Bortezomib (1.3 mg/m2) and temozolomide (75 mg/m2) proved to be the maximum tolerated dose. Dose-limiting toxicities included neurotoxicity, fatigue, diarrhea, and rash. Nineteen melanoma patients were enrolled onto four dose levels. This melanoma population (17 M1c, 10 elevated lactate dehydrogenase, 12 performance status 1-2) showed only one partial response (8 months) and three with stable disease ≥4 months. A significant reduction in proteasome-specific activity was observed 1 hour after infusion at all bortezomib doses. Changes in NF-κB electrophoretic mobility shift assay and circulating chemokines in blood failed to correlate with the schedule/dose of bortezomib, inhibition of proteasome activity, or clinical outcome.Conclusions: We have defined phase II doses for this schedule of temozolomide with bortezomib. Although proteasome activity was inhibited for a limited time in peripheral blood mononuclear cells, we were unable to show consistent effects on NF-κB activation. Clin Cancer Res; 16(1); 348–57

Supplementary Data from A Phase I Trial of Bortezomib with Temozolomide in Patients with Advanced Melanoma: Toxicities, Antitumor Effects, and Modulation of Therapeutic Targets

Supplementary Table S1. A. NGS Summary.

Purpose:Pancreatic cancer is among the most aggressive malignancies and is rarely discovered early. However, pancreatic “incidentalomas,” particularly cysts, are frequently identified in asymptomatic patients through anatomic imaging for unrelated causes. Accurate determination of the malignant potential of cystic lesions could lead to life-saving surgery or spare patients with indolent disease undue risk. Current risk assessment of pancreatic cysts requires invasive sampling, with attendant morbidity and sampling errors. Here, we sought to identify imaging biomarkers of high-risk pancreatic cancer precursor lesions.Experimental Design:Translocator protein (TSPO) expression, which is associated with cholesterol metabolism, was evaluated in premalignant and pancreatic cancer lesions from human and genetically engineered mouse (GEM) tissues. In vivo imaging was performed with [18F]V-1008, a TSPO-targeted PET agent, in two GEM models. For image-guided surgery (IGS), V-1520, a TSPO ligand for near-IR optical imaging based upon the V-1008 pharmacophore, was developed and evaluated.Results:TSPO was highly expressed in human and murine pancreatic cancer. Notably, TSPO expression was associated with high-grade, premalignant intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanIN) lesions. In GEM models, [18F]V-1008 exhibited robust uptake in early pancreatic cancer, detectable by PET. Furthermore, V-1520 localized to premalignant pancreatic lesions and advanced tumors enabling real-time IGS.Conclusions:We anticipate that combined TSPO PET/IGS represents a translational approach for precision pancreatic cancer care through discrimination of high-risk indeterminate lesions and actionable surgery.

Supplementary Figure 1: BKM120 inhibited CD4+ T cell, not CD8+ T cell proliferation and had small effects on PyMT specific T cell mediated cytotoxicity only at a 1:50 E/T cell ratio. Supplementary Figure 2: Tumors were initiated as described in Figure 1 and BKM120 was delivered to PyMT-tumor bearing C57Bl/6 mice (n = 5) for different periods of time. Supplementary Figure 3C and 3D. Additional Flow Cytometry Analysis of Leukocyte populations in PD-1 treated animals. Supplementary Figure 3E and 3F. Myeloid FACS analysis and FoxP3 histology from BKM-120 and �PD-1 treated PyMT Supplemental Figure 5: Implantation of expanded human T cells into NSG mice. Supplemental Figure 7: Effects of Loss of PI3Kγ on Leukocytes

Supplementary Data from Identification of Markers of Taxane Sensitivity Using Proteomic and Genomic Analyses of Breast Tumors from Patients Receiving Neoadjuvant Paclitaxel and Radiation

Purpose:Over 60% of patients with melanoma respond to immune checkpoint inhibitor (ICI) therapy, but many subsequently progress on these therapies. Second-line targeted therapy is based on BRAF mutation status, but no available agents are available for NRAS, NF1, CDKN2A, PTEN, and TP53 mutations. Over 70% of melanoma tumors have activation of the MAPK pathway due to BRAF or NRAS mutations, while loss or mutation of CDKN2A occurs in approximately 40% of melanomas, resulting in unregulated MDM2-mediated ubiquitination and degradation of p53. Here, we investigated the therapeutic efficacy of over-riding MDM2-mediated degradation of p53 in melanoma with an MDM2 inhibitor that interrupts MDM2 ubiquitination of p53, treating tumor-bearing mice with the MDM2 inhibitor alone or combined with MAPK-targeted therapy.Experimental Design:To characterize the ability of the MDM2 antagonist, KRT-232, to inhibit tumor growth, we established patient-derived xenografts (PDX) from 15 patients with melanoma. Mice were treated with KRT-232 or a combination with BRAF and/or MEK inhibitors. Tumor growth, gene mutation status, as well as protein and protein–phosphoprotein changes, were analyzed.Results:One-hundred percent of the 15 PDX tumors exhibited significant growth inhibition either in response to KRT-232 alone or in combination with BRAF and/or MEK inhibitors. Only BRAFV600WT tumors responded to KRT-232 treatment alone while BRAFV600E/M PDXs exhibited a synergistic response to the combination of KRT-232 and BRAF/MEK inhibitors.Conclusions:KRT-232 is an effective therapy for the treatment of either BRAFWT or PANWT (BRAFWT, NRASWT) TP53WT melanomas. In combination with BRAF and/or MEK inhibitors, KRT-232 may be an effective treatment strategy for BRAFV600-mutant tumors.

Supplementary Information including Supplementary Tables S1, S2 and S3 and Legends for Supplementary Movies S1 and S2

Real-time image-guided surgery of Ptf1a-Cre;LSL-KrasG12D/+;Smad4fl/fl (KSC) mouse using V-1520

Purpose: Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,β in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered antitumor immunity.Experimental Design: The effect of PI3K inhibition on tumor growth, metastasis, and antitumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ-null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor-infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30 mg/kg, every day) or vehicle-treated mice and PI3Kγnull versus PI3KγWT mice. On the basis of the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30 mg/kg, every day) and anti-PD1 (100 μg, twice weekly) was evaluated in PyMT tumor–bearing mice.Results: Our findings show that PI3K activity facilitates tumor growth and surprisingly restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The antitumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+ T-cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared with either agent alone.Conclusions: PI3K inhibition slows tumor growth, enhances antitumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple-negative breast cancer. Clin Cancer Res; 23(13); 3371–84. ©2016 AACR.

Purpose: To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies.Experimental Design: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR).Results: Proteomic and validation immunohistochemical analyses revealed that α-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery.Conclusion: We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane–based therapy. Clin Cancer Res; 16(2); 681–90

Supplementary Methods. Description of additional methods and procedures used in the study.

PDF file, 308K, Supplemental Figure S1. Tumoral IL-6 promotes angiosarcoma growth. Supplemental Figure S2. IL-6 triggers Stat3 signal pathway and impacts tumor immunity. Supplemental Figure S3. Immune cell migration is Ikkβ dependent and IL-6 independent. Supplemental Figure S4. Deletion of Ikk in myeloid cells impairs myeloid migration in response to tumor in vivo. Supplemental Figure S5. Systemic targeting of Ikkβ/Stat3/IL-6 in vivo. Supplemental Figure S7. Tumor induced myeloid cell chemoattraction assay in vivo. Supplemental Table S1. Myeloid IKKβ impacts angiosarcoma incidence and latency. Supplemental Table S2. Alignment of κB sequence from murine gp130, JAK2 and IL-6 promoters.

Supplementary Methods

Figure S1: Myeloid Ikkβ is required for anti-tumorigenic immunity. Figure S2. Myeloid cell depletion alters tumor immunity. Figure S3. Ikkβ loss alters macrophage phagocytosis capacity. Figure S4. Myeloid Ikkβ deletion reduced the cytotoxicity of T cells Figure S5. Cytokines play a major role in orchestrating the cellular make-up of the tumor microenvironment.

Supplementary Figure S7. Results of the flow cytometric analysis of indicated populations of leukocytes in SK-Mel5 tumors grown in BALB/C nu/Foxn1 athymic nude mice.

Supplementary Table S1. Parameter estimates and interindividual variability for the (�)�Nutlin�3 pharmacokinetic model.

PDF file, 154K.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Leveraging spatial molecular information to construct a phylogeographic map of tumor evolution can reveal individualized growth trajectories with diagnostic and therapeutic potential. Integrative analysis of spatial multi-omic data from 31 colorectal specimens revealed simultaneous microenvironmental and clonal alterations as a function of progression. Copy number variation served to re-stratify microsatellite stable and unstable tumors into chromosomally unstable (CIN+) and hypermutated (HM) classes. Phylogeographical maps classified tumors by their evolutionary dynamics, and clonal regions were placed along a global pseudotemporal progression trajectory. Cell-state discovery from a single-cell cohort revealed recurring epithelial gene signatures and infiltrating immune states in spatial transcriptomics. Charting these states along progression pseudotime, we observed a transition to immune exclusion in CIN+ tumors as characterized by a novel gene expression signature comprised ofDDR1, TGFBI, PAK4,andDPEP1. We demonstrated how these genes and their protein products are key regulators of extracellular matrix components, are associated with lower cytotoxic immune infiltration, and show prognostic value in external cohorts. Through high-dimensional data integration, this atlas provides insights into co-evolution of tumors and their microenvironments, serving as a resource for stratification and targeted treatment of CRC.

BACKGROUND: Back pain is a leading reason for seeking care in the United States (US), and is a major cause of morbidity. OBJECTIVE: To analyze demographic, patient, and visit characteristics of adult ambulatory spine clinic visits in the United States from 2009–2016. METHODS: Data from the National Ambulatory Medical Care Survey from 2009–2016 were used and were sample weighted. RESULTS: Most patients presenting for ambulatory spine care were 45–64 years (45%), were most commonly female (56.8%), and private insurance (45%) and Medicare (26%) were most common payors. The percentage of visits for spine care done at a primary care setting was 50.1% in 2009–2010 and 48.3% in 2014–2015. Approximately 15.5% were seen in orthopedic surgery clinics in 2009–2010 and 7.3% in 2015–2016. MRI was utilized in 11.7% in 2009–2010 and 11.0% in 2015–2016. Physical therapy was prescribed in 13.2% and narcotic analgesic medications were prescribed in 36.2% of patients in 2015–2016. CONCLUSIONS: MRI was used more frequently than guidelines recommended, and physical therapy was less frequently utilized despite evidence. A relatively high use of opiates in treatment of back pain was reported and is concerning. Although back pain represents a substantial public health burden in the United States, the delivery of care is not evidence-based.

Background While immune checkpoint blockade (ICB) is the current first-line treatment for metastatic melanoma, it is effective for ~ 52% of patients and has dangerous side effects. The objective here was to identify the feasibility and mechanism of RAS/RAF/PI3K pathway inhibition in melanoma to sensitize tumors to ICB therapy. Methods Rigosertib (RGS) is a non-ATP-competitive small molecule RAS mimetic. RGS monotherapy or in combination therapy with ICB were investigated using immunocompetent mouse models of BRAFwt and BRAFmut melanoma and analyzed in reference to patient data. Results RGS treatment (300 mg/kg) was well tolerated in mice and resulted in ~ 50% inhibition of tumor growth as monotherapy and ~ 70% inhibition in combination with αPD1 + αCTLA4. RGS-induced tumor growth inhibition depends on CD40 upregulation in melanoma cells followed by immunogenic cell death, leading to enriched dendritic cells and activated T cells in the tumor microenvironment. The RGS-initiated tumor suppression was partially reversed by either knockdown of CD40 expression in melanoma cells or depletion of CD8+ cytotoxic T cells. Treatment with either dabrafenib and trametinib or with RGS, increased CD40+SOX10+ melanoma cells in the tumors of melanoma patients and patient-derived xenografts. High CD40 expression level correlates with beneficial T-cell responses and better survival in a TCGA dataset from melanoma patients. Expression of CD40 by melanoma cells is associated with therapeutic response to RAF/MEK inhibition and ICB. Conclusions Our data support the therapeutic use of RGS + αPD1 + αCTLA4 in RAS/RAF/PI3K pathway-activated melanomas and point to the need for clinical trials of RGS + ICB for melanoma patients who do not respond to ICB alone. Trial registration NCT01205815 (Sept 17, 2010). Graphical abstract