Person:

Rodig, Scott

There is a pressing need for methods to define the functional relevance of genetic alterations identified by next-generation sequencing of cancer specimens. We developed new approaches to efficiently construct full-length cDNA libraries from small amounts of total RNA, screen for transforming and resistance phenotypes, and deconvolute by next-generation sequencing. Using this platform, we screened a panel of cDNA libraries from primary specimens and cell lines in cytokine-dependent murine Ba/F3 cells. We demonstrate that cDNA library-based screening can efficiently identify DNA and RNA alterations that confer either cytokine-independent proliferation or resistance to targeted inhibitors, including RNA alterations and intergenic fusions. Using barcoded next-generation sequencing, we simultaneously deconvoluted cytokine-independent clones recovered after transduction of 21 cDNA libraries. This approach identified multiple gain-of-function alleles, including KRAS G12D, NRAS Q61K and an activating splice variant of ERBB2. This approach has broad applicability for identifying transcripts that confer proliferation, resistance and other phenotypes in vitro and potentially in vivo.

OBJECTIVES—To investigate a B-cell–depleting strategy to reverse diabetes in naïve NOD mice. RESEARCH DESIGN AND METHODS—We targeted the CD22 receptor on B-cells of naïve NOD mice to deplete and reprogram B-cells to effectively reverse autoimmune diabetes. RESULTS—Anti-CD22/cal monoclonal antibody (mAb) therapy resulted in early and prolonged B-cell depletion and delayed disease in pre-diabetic mice. Importantly, when new-onset hyperglycemic mice were treated with the anti-CD22/cal mAb, 100% of B-cell–depleted mice became normoglycemic by 2 days, and 70% of them maintained a state of long-term normoglycemia. Early therapy after onset of hyperglycemia and complete B-cell depletion are essential for optimal efficacy. Treated mice showed an increase in percentage of regulatory T-cells in islets and pancreatic lymph nodes and a diminished immune response to islet peptides in vitro. Transcriptome analysis of reemerging B-cells showed significant changes of a set of proinflammatory genes. Functionally, reemerging B-cells failed to present autoantigen and prevented diabetes when cotransferred with autoreactive (CD4^+) T-cells into NOD.SCID hosts. CONCLUSIONS—Targeting CD22 depletes and reprograms B-cells and reverses autoimmune diabetes, thereby providing a blueprint for development of novel therapies to cure autoimmune diabetes.

Background: Anaplastic lymphoma kinase (ALK) genomic alterations have emerged as a potent predictor of benefit from treatment with ALK inhibitors in several cancers. Currently, there is no information about ALK gene alterations in urothelial carcinoma (UC) and its correlation with clinical or pathologic features and outcome. Methods: Samples from patients with advanced UC and correlative clinical data were collected. Genomic imbalances were investigated by array comparative genomic hybridization (aCGH). ALK gene status was evaluated by fluorescence in situ hybridization (FISH). ALK expression was assessed by immunohistochemistry (IHC) and high-throughput mutation analysis with Oncomap 3 platform. Next generation sequencing was performed using Illumina Genome Analyzer IIx, and Illumina HiSeq 2000 in the FISH positive case. Results: 70 of 96 patients had tissue available for all the tests performed. Arm level copy number gains at chromosome 2 were identified in 17 (24%) patients. Minor copy number alterations (CNAs) in the proximity of ALK locus were found in 3 patients by aCGH. By FISH analysis, one of these samples had a deletion of the 5′ALK. Whole genome next generation sequencing was inconclusive to confirm the deletion at the level of the ALK gene at the coverage level used. We did not observe an association between ALK CNA and overall survival, ECOG PS, or development of visceral disease. Conclusions: ALK genomic alterations are rare and probably without prognostic implications in UC. The potential for testing ALK inhibitors in UC merits further investigation but might be restricted to the identification of an enriched population.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.

Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients.

Although the treatment of acute myeloid leukemia (AML) has improved significantly, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified aberrant expression of the hepatocyte growth factor (HGF) as a critical factor in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance due to compensatory upregulation of HGF expression, leading to restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked compensatory HGF upregulation, resulting in sustained logarithmic cell kill both in vitro and in xenograft models in vivo. Our results demonstrate widespread dependence of AML cells on autocrine activation of MET, as well as the importance of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

Importance Immune checkpoint inhibitor therapy has shown benefit in various cancers, but their potential in endometrial cancer (EC) is unknown.

Observations Prediction of neoantigen load was performed using sequencing data from the Cancer Genome Atlas data set. Evaluation of tumor-infiltrating lymphocytes (TILs) and PD-1 and PD-L1 expression was performed in 63 patients with EC referred to our institution. The predicted median (range) neoantigen load (predicted neoepitopes per sample) was proportional to the mutational load: highest in ultramutated polymerase e (POLE) tumors (8342 [628-20 440]), less in hypermutated MSI (541 [146-8063]; P < .001), and lowest in microsatellite-stable tumors (70.5 [7-1877]; P < .001). The POLE and MSI ECs exhibited higher numbers of CD3+ (44.5 vs 21.8; P = .001) and CD8+ (32.8 vs 13.5; P < .001) TILs compared with microsatellite-stable tumors. PD-1 was overexpressed in TILs (81% vs 28%; P < .001) and peritumoral lymphocytes (90% vs 28%; P < .001) of POLE and MSI tumors. PD-L1 expression was infrequently noted in tumor cells but was common in intraepithelial immune cells and more frequent in POLE and MSI tumors (39% vs 13%; P = .02).

Conclusions and Relevance Polymerase e–mutated and MSI ECs are associated with high neoantigen loads and number of TILs, which is counterbalanced by overexpression of PD-1 and PD-L1. Polymerase e–mutated and MSI EC tumors may be excellent candidates for PD-1–targeted immunotherapies.

ROS1 gene rearrangements are reported in 1–2% of lung adenocarcinomas (ACA) and are associated with response to the multitargeted tyrosine kinase inhibitor, crizotinib. ROS1 rearrangements can be detected using fluorescence in situ hybridization (FISH) however immunohistochemistry (IHC) for ROS1 protein is a promising alternate screening modality. In this study we examine the correlation between ROS1 IHC and FISH and describe the clinicopathologic characteristics of ROS1-rearranged lung tumors. ROS1 IHC was performed using clone D4D6 (Cell Signaling Technology, Danvers, MA) on whole tissue sections. In a validation cohort, IHC was compared to ROS1 break-apart FISH in 53 cases of lung ACA enriched for an absence of known genetic alterations and never-smoking status. In a screening cohort, we performed ROS1 IHC on 167 consecutive cases of lung ACA from a routine molecular diagnostics practice and confirmed positive results by FISH. In the validation cohort, 6 cases (11%) were both FISH and IHC positive. One FISH-negative case was strongly ROS1 IHC positive. All IHC negative cases were FISH negative. In the screening cohort, 2 of 167 (1.2%) had strong, diffuse ROS1 protein expression; a rearrangement was confirmed by FISH in both. ROS1-translocated tumors were wild type for EGFR, KRAS, and ALK and commonly had solid growth with mucinous/cribriform features and psammomatous calcification. ROS1 protein expression in tumor cells is 100% sensitive and 92% specific for ROS1 rearrangements by FISH. ROS1 IHC is an effective screening tool for this rare but clinically important subset of lung ACA.