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Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions

Tocheva, Anna S; Peled, Michael; Strazza, Marianne; Adam, Kieran R; Lerrer, Shalom; Nayak, Shruti; Azoulay-Alfaguter, Inbar; Foster, Connor J R; Philips, Elliot A; Neel, Benjamin; Ueberheide, Beatrix; Mor, Adam
Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell mediated anti-tumor immunity but many patients do not respond and a significant proportion develops inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1 triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.
PMID: 33077516
ISSN: 1083-351x
CID: 4642072

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε

Sviderskiy, Vladislav O; Blumenberg, Lili; Gorodetsky, Elizabeth; Karakousi, Triantafyllia R; Hirsh, Nicole; Alvarez, Samantha W; Terzi, Erdem M; Kaparos, Efiyenia; Whiten, Gabrielle C; Ssebyala, Shakirah; Tonzi, Peter; Mir, Hannan; Neel, Benjamin G; Huang, Tony T; Adams, Sylvia; Ruggles, Kelly V; Possemato, Richard
Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.
PMID: 33152268
ISSN: 1097-4164
CID: 4664322

Dual genetic targeting of ERK1 and ERK2 reduces the fitness of the malignant clone in myeloproliferative neoplasm mice [Meeting Abstract]

Stivala, S; Brkic, S; Santopolo, A; Dirnhofer, S; Passweg, J R; Tsakiris, D; Neel, B; Levine, R L; Meyer, S C
Introduction: Myeloproliferative neoplasms (MPN) are chronic leuke-mias with dysregulated Jak2 signaling. We hypothesized that genetic tar-geting of ERK1/2 could enhance control of the MPN clone by preventing MAPK pathway activation.
Method(s): We genetically targeted ERK1/2 in MPN by introducing ERK1/2 knockout alleles and hematopoiesis-specific Mx-Cre in Jak2V617F mice. To assess engraftment and competitive fitness of the MPN clone, CD45.2 Jak2V617F bone marrow (BM) +/- ERK1-/- ERK2fl/fl was competitively transplanted with CD45.1 Jak2 WT BM.
Result(s): Loss of ERK1/2 in Jak2V617F Ba/F3 cells reduced cells growth by 60% and potentiated Jak2 inhibition by ruxolitinib. In MPN mice, it moderated splenomegaly and excessive erythropoiesis including red cells, reticulocytes and erythroid progenitors. Hematopoietic stem/pro-genitor compartments were reduced and myeloid colony formation di-minished in Jak2V617F ERK1-/- ERK2fl/fl mice, suggesting reduced dis-ease-initiating cells. In competitive transplants, ERK1/2 loss significantly reduced the Jak2V617F MPN clone in peripheral blood, BM, myeloid and erythroid progenitors. Myeloid colonies emerging from Jak2V617F ERK1-/- ERK2fl/fl:WT competitively transplanted mice were predominantly Jak2 WT as compared to settings with intact ERK. Polyglobulia was nor-malized and BM fibrosis prevented in recipients of Jak2V617F ERK1-/- ERK2fl/fl BM. ERK1/2 deletion combined with Jak2 inhibition with rux-olitinib enhanced therapeutic efficacy with extensive reduction of the MPN clone and correction of the MPN phenotype.
Conclusion(s): ERK1/2 loss abrogates the competitive fitness of the MPN clone by restricting stem/progenitor compartments and cooperates with JAK2 inhibition resulting in correction of MPN features. Our data suggest targeting of ERK1/ERK2 in combination with JAK2 inhibition as an en-hanced therapeutic strategy in MPN
ISSN: 1424-3997
CID: 4757452

U.S. Biomedical Research Needs More Immigrant Scientists, Not Fewer! [Letter]

Aifantis, Iannis; Neel, Benjamin G
PMID: 32931738
ISSN: 1878-3686
CID: 4592912

Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21

Hussain, Ali; Voisin, Veronique; Poon, Stephanie; Karamboulas, Christina; Bui, Ngoc Hoang Bao; Meens, Jalna; Dmytryshyn, Julia; Ho, Victor W; Tang, Kwan Ho; Paterson, Joshua; Clarke, Blaise A; Bernardini, Marcus Q; Bader, Gary D; Neel, Benjamin G; Ailles, Laurie E
Recent studies indicate that cancer-associated fibroblasts (CAFs) are phenotypically and functionally heterogeneous. However, little is known about CAF subtypes, the roles they play in cancer progression, and molecular mediators of the CAF "state." Here, we identify a novel cell surface pan-CAF marker, CD49e, and demonstrate that two distinct CAF states, distinguished by expression of fibroblast activation protein (FAP), coexist within the CD49e+ CAF compartment in high-grade serous ovarian cancers. We show for the first time that CAF state influences patient outcomes and that this is mediated by the ability of FAP-high, but not FAP-low, CAFs to aggressively promote proliferation, invasion and therapy resistance of cancer cells. Overexpression of the FAP-low-specific transcription factor TCF21 in FAP-high CAFs decreases their ability to promote invasion, chemoresistance, and in vivo tumor growth, indicating that it acts as a master regulator of the CAF state. Understanding CAF states in more detail could lead to better patient stratification and novel therapeutic strategies.
PMID: 32434219
ISSN: 1540-9538
CID: 4444422

Genetic aberrations dictate distinct tumor immune landscape and chemosensitivity in HGSOC [Meeting Abstract]

Zhang, S; Iyer, S; Ran, H; Wei, W; Weinberg, R A; Neel, B G
High-grade serous ovarian cancer (HGSOC) is the most common and deadly subtype of ovarian epithelial cancer and is known for its aggressiveness, high recurrence rate, metastasis to other sites, development of resistance to conventional chemotherapy, and general lack of response to immune checkpoint inhibitors. The absence of genomically relevant, immune-competent HGSOC models represents a major barrier to developing new therapies. Taking advantage of a mouse fallopian tube organoid system that we developed, along with lentiviral gene transduction and/or CRISPR/Cas9 technology, we generated multiple new HGSOC models containing combinations of mutations seen in human HGSOC, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1amp;Akt2ampand Tp53-/-;Ccne1amp;Krasamp) and -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-;Brca1-/-;Mycamp), and poorly characterized (Tp53-/-;Pten-/-;Nf1-/-) models. These models differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenic capacity and behavior upon orthotopic injection into syngeneic mice. Organoids bearing different mutational spectra show differential sensitivity to conventional HGSOC chemotherapies, signal transduction inhibitors and DDR inhibitors, and evoke distinctly different immune microenvironment in vivo. In particular, the immune microenvironment induced by HRdeficient tumors shows more T-cell infiltration/Treg cells, whereas HR-proficient lines show lower T-cell infiltration but higher levels of myeloid-derived suppressor cells and macrophages. The results of these studies suggest novel, genotype-informed combination therapies for this devastating disease
ISSN: 1557-3265
CID: 4659082

The genotype of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immune checkpoint therapies [Meeting Abstract]

Iyer, S; Zhang, S; Farkkila, A; Smith, S; Pepin, D; Mohan, R; Xia, T; Reinhardt, F; Chavarria, T; Hoefsmit, E; Poort, V; Pathania, S; Zhou, Y; Elias, K M; Hammond, P T; Neel, B G; Weinberg, R A
Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) DNA repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombinationdeficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNASeq (scRNASeq) transcriptomic, and immunofluorescence imaging analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease
ISSN: 1557-3265
CID: 4659102

Raymond L. Erikson (1936-2020)

Neel, Benjamin G; Sweet, Laurel; Simmons, Daniel L; Blenis, John
PMID: 33074798
ISSN: 1097-4172
CID: 4641972

Piecing Together a Broken Tumor Suppressor Phosphatase for Cancer Therapy

Westermarck, Jukka; Neel, Benjamin G
Members of the PP2A family of serine/threonine phosphatases are important human tumor suppressor genes. Unlike most tumor suppressors, they are rarely mutated/deleted, but rather are impaired by "inhibitor proteins." Two papers in this issue of Cell show how some phenothiazine derivatives reactivate specific PP2A isozymes with potential benefit in cancer and other diseases.
PMID: 32359434
ISSN: 1097-4172
CID: 4415702

The sixth international RASopathies symposium: Precision medicine-From promise to practice

Gripp, Karen W; Schill, Lisa; Schoyer, Lisa; Stronach, Beth; Bennett, Anton M; Blaser, Susan; Brown, Amanda; Burdine, Rebecca; Burkitt-Wright, Emma; Castel, Pau; Darilek, Sandra; Dias, Alwyn; Dyer, Tuesdi; Ellis, Michelle; Erickson, Gregg; Gelb, Bruce D; Green, Tamar; Gross, Andrea; Ho, Alan; Holder, James Lloyd; Inoue, Shin-Ichi; Jelin, Angie C; Kennedy, Annie; Klein, Richard; Kontaridis, Maria I; Magoulas, Pilar; McConnell, Darryl B; McCormick, Frank; Neel, Benjamin G; Prada, Carlos E; Rauen, Katherine A; Roberts, Amy; Rodriguez-Viciana, Pablo; Rosen, Neal; Rumbaugh, Gavin; Sablina, Anna; Solman, Maja; Tartaglia, Marco; Thomas, Angelica; Timmer, William C; Venkatachalam, Kartik; Walsh, Karin S; Wolters, Pamela L; Yi, Jae-Sung; Zenker, Martin; Ratner, Nancy
The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.
PMID: 31825160
ISSN: 1552-4833
CID: 4234482