Amit Deshpande

Cyclins and cdks in development and cancer: a perspective

A fundamental aspect of cancer is dysregulated cell cycle control. Unlike normal cells that only proliferate when compelled to do so by developmental or other mitogenic signals in response to tissue growth needs, the proliferation of cancer cells proceeds essentially unchecked. This does not mean that cancer cell cycles are necessarily different from those found in normal cycling cells, but rather implies that cancer cells proliferate because they are no longer subject to proliferation-inhibitory influences arising from the stroma or from gene expression pattern changes consequent to ‘terminal’ differentiation, nor do they necessarily require extrinsic growth factors to recruit them into or maintain their proliferative state. Finally, cancer cells have also often avoided normal controls linked to cell cycle progression that halt proliferation in the presence of damaged DNA or other physiological insults. The result of these alterations is the inappropriate proliferation commonly associated with cancerous tumor formation. This review will summarize the current understanding of dysregulation of the G0/G1-to-S-phase transition in cancer cells, with particular emphasis on recent in vivo studies that suggest a need to rethink existing models of cell cycle control in development and tumorigenesis.

A Requirement for Cyclin-Dependent Kinase 6 in Thymocyte Development and Tumorigenesis

Cyclin-dependent kinase 6 (CDK6) promotes cell cycle progression and is overexpressed in human lymphoid malignancies. To determine the role of CDK6 in development and tumorigenesis, we generated and analyzed knockout mice. Cdk6-deficient mice show pronounced thymic atrophy due to reduced proliferative fractions and concomitant transitional blocks in the double-negative stages. Using the OP9-DL1 system to deliver temporally controlled Notch receptor-dependent signaling, we show that CDK6 is required for Notch-dependent survival, proliferation, and differentiation. Furthermore, CDK6-deficient mice were resistant to lymphomagenesis induced by active Akt, a downstream target of Notch signaling. These results show a critical requirement for CDK6 in Notch/Akt-dependent T-cell development and tumorigenesis and strongly support CDK6 as a specific therapeutic target in human lymphoid malignancies.

The retinoblastoma protein in osteoblast differentiation and osteosarcoma.

Osteogenic sarcoma (osteosarcoma) is the most common primary tumor of bone. It accounts for approximately 19% of all malignant tumors of the bone. Of all the molecular targets altered during the genesis of osteosarcoma, the retinoblastoma gene (RB1) shows the highest frequency of inactivation. Published data from human osteosarcoma tumors and in vivo and in vitro model systems support a role for the retinoblastoma gene family in bone development and tumorigenesis. Although a variety of bone tumors, depending on the cell of origin, including osteoclasts or osteoclast-like cells, chondroblasts, and fibroblasts, are described, for the purpose of this review we will focus primarily on the tumors arising from the osteoblast lineage.

PHC3, a component of the hPRC-H complex, associates with E2F6 during G0 and is lost in osteosarcoma tumors

Polyhomeotic-like 3 (PHC3) is a ubiquitously expressed member of the polycomb gene family and part of the human polycomb complex hPRC-H. We found that in normal cells PHC3 associated with both hPRC-H complex components and with the transcription factor E2F6. In differentiating and confluent cells, PHC3 and E2F6 showed nuclear colocalization in a punctate pattern that resembled the binding of polycomb bodies to heterochromatin. This punctate pattern was not seen in proliferating cells suggesting that PHC3 may be part of an E2F6-polycomb complex that has been shown to occupy and silence target promoters in G0. Previous loss of heterozygosity (LoH) analyses had shown that the region containing PHC3 underwent frequent LoH in primary human osteosarcoma tumors. When we examined normal bone and human osteosarcoma tumors, we found loss of PHC3 expression in 36 of 56 osteosarcoma tumors. Sequence analysis revealed that PHC3 was mutated in nine of 15 primary osteosarcoma tumors. These findings suggest that loss of PHC3 may favor tumorigenesis by potentially disrupting the ability of cells to remain in G0.

CDK6 kinase activity is required for thymocyte development

Cyclin-dependent kinase-6 (CDK6) is required for early thymocyte development and tumorigenesis. To mechanistically dissect the role of CDK6 in thymocyte development, we generated and analyzed mutant knock-in mice and found that mice expressing a kinase-dead Cdk6 allele (Cdk6(K43M)) had a pronounced reduction in thymocytes and hematopoietic stem cells and progenitor cells (Lin⁻Sca-1⁺c-Kit⁺ [LSK]). In contrast, mice expressing the INK4-insensitive, hyperactive Cdk6(R31C) allele displayed excess proliferation in LSK and thymocytes. However, this is countered at least in part by increased apoptosis, which may limit progenitor and thymocyte expansion in the absence of other genetic events. Our mechanistic studies demonstrate that CDK6 kinase activity contributes to Notch signaling because inactive CDK6 kinase disrupts Notch-dependent survival, proliferation, and differentiation of LSK, with concomitant alteration of Notch target gene expression, such as massive up-regulation of CD25. Further, knockout of CD25 in Cdk6(K43M) mice rescued most defects observed in young mice. These results illustrate an important role for CDK6 kinase activity in thymocyte development that operates partially through modulating Notch target gene expression. This role of CDK6 as a downstream mediator of Notch identifies CDK6 kinase activity as a potential therapeutic target in human lymphoid malignancies.

CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1- induced T cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy.

Abstract 573: Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody

ICOS (inducible co-stimulator molecule) is a co-stimulatory molecule and a member of the CD28 superfamily expressed primarily on T lymphocytes. Analysis of cancer patient samples as well as rodent preclinical data have implicated a role for the ICOS pathway in cancer immunotherapy. We have generated a panel of anti-ICOS monoclonal antibodies with in vitro agonistic properties. The anti-ICOS antibodies are efficacious as monotherapies and in combination with anti-PD1 in multiple syngeneic tumor models. Mechanistic studies demonstrate that tumor regression is associated with enhanced ratios of cytotoxic CD8:T regulatory (Treg) cells as well as preferential reduction in ICOS-high Tregs in the tumor microenvironment. JTX-2011, a species cross-reactive high affinity humanized agonist monoclonal antibody, has been selected for development. Evaluation of JTX-2011 in nonhuman primate models will be presented, including data informing safety and PK parameters. Our preclinical data provides rational for clinical development of JTX-2011 as a cancer immunotherapeutic to be tested as both a monotherapy as well as in combination with immunotherapies in solid tumor indications. Citation Format: Jennifer S. Michaelson, Christopher J. Harvey, Kutlu G. Elpek, Ellen Duong, Matthew Wallace, Chengyi J. Shu, Sriram Sathyanarayanan, Robert Mabry, Lindsey Shallberg, Tong Zi, Amit Deshpande, Stephen L. Sazinsky, Joshua Apgar, Deborah Law. Preclinical evaluation of JTX-2011, an anti-ICOS agonist antibody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 573.

Abstract 5446: FPA144: A therapeutic antibody for treating patients with gastric cancers bearing FGFR2 gene amplification

A subset of patients with gastric cancer have an amplification of the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) gene. The amplification is most common in the diffuse type of gastric cancer and its presence correlates with poor patient prognosis. Although it has been reported that there is high expression of FGFR2 protein in patients with the amplification, it has been unknown which of the two major FGFR2 isoforms, FGFR2b or 2c, is expressed. In this study, we demonstrate, by both qPCR and immunohistochemistry using FGFR2b-selective antibodies, that it is the FGFR2b isoform, and not FGFR2c, that is overexpressed in gastric cancer tumors that contain the FGFR2 amplification. Five Prime Therapeutics has developed an FGFR2b-specific antibody, FPA144, to treat patients with gastric cancers bearing amplification of the FGFR2 gene. FPA144 is glycoengineered for enhanced antibody-dependent cell cytotoxicity (ADCC). FPA144 causes tumor growth inhibition by 72% to 100% in gastric cancer xenograft models with FGFR2 gene amplification that overexpress the FGFR2b protein isoform. In the SNU-16 model, FPA144 reduces the levels of FGFR2b protein expressed in the tumors by approximately 50%, and decreases both FGFR2b phosphorylation and phosphorylation of the downstream effector, FRS2. The anti-tumor effect of FPA144 is additive with the standard of care chemotherapy regimens of 5-fluoruracil/cisplatin and paclitaxel in the OCUM-2 gastric cancer xenograft model. Since FPA144, unlike small molecule FGFR tyrosine kinase inhibitors, blocks signaling by only the FGFR2b and not the other FGFRs, we expect a favorable toxicology profile, either alone or in combination with chemotherapy. In addition, by engaging immune effector cells for direct tumor cell killing, FPA144 has a second mechanism of action that small molecule FGFR tyrosine kinase inhibitors do not have. We anticipate starting clinical trials of FPA144 in the last quarter of 2014. Citation Format: Abigael T. Gemo, Amit M. Deshpande, Servando Palencia, David I. Bellovin, Thomas J. Brennan, Namrata S. Patil, Carol Huang, Gerrit Los, Kristen L. Pierce. FPA144: A therapeutic antibody for treating patients with gastric cancers bearing FGFR2 gene amplification. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5446. doi:10.1158/1538-7445.AM2014-5446

Abstract SY03-02: Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial

ICOS (the inducible T-cell co-stimulator) is a co-stimulatory molecule expressed on the surface of T cells and a member of the CD28 family, which includes clinically validated targets of cancer immunotherapies, such as PD-1 and CTLA-4. Clinical data identified ICOS as a potentially key molecule in providing optimal antitumor benefit following anti-CTLA-4 therapy. We have developed a species cross-reactive humanized IgG1 agonist antibody, JTX-2011, that binds ICOS and is designed to induce an antitumor immune response. Our preclinical data suggest that JTX-2011 functions through a dual mechanism of action, by stimulating T effector cells (Teff) and depleting intratumoral T regulatory cells (Tregs). The ICOS antibody is efficacious as a single agent in mouse syngeneic tumor models and demonstrates enhanced activity when administered in combination with anti-PD-1. Single-agent activity in the preclinical models appears to correlate with ICOS expression, with greater efficacy observed in tumor models that exhibit a higher percentage of ICOS-expressing immune cell infiltrate. An integrated expression analysis of human tumors identified non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) as indications with higher percentages of ICOS-expressing cell infiltrate. Preclinical studies performed in rodent and monkeys evaluated safety, pharmacokinetics, and pharmacodynamics of JTX-2011 to inform the first in-human study. The ICONIC phase I/II clinical trial is currently ongoing for evaluation of JTX-2011 alone or in combination with the anti-PD-1 antibody Nivolumab in patients with advanced solid tumors and incorporates a patient enrichment strategy design based on the preclinical and translational findings. Citation Format: Jennifer S. Michaelson, Christopher Harvey, Kutlu Elpek, Ellen Duong, Lindsey Shallberg, Matthew Wallace, Robert Mabry, Jenny Shu, Amit Deshpande, Tong Zi, Stephen Sazinsky, Joshua Apgar, Barbara Mounho-Zamora, Michael Briskin, Elizabeth Trehu, Jason Reeves, Heather Hirsch, Sriram Sathyanarayanan, Deborah Law. Preclinical assessment of JTX-2011, an agonist antibody targeting ICOS, supports evaluation in ICONIC clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr SY03-02. doi:10.1158/1538-7445.AM2017-SY03-02

Abstract A052: Characterization of immune regulatory molecules B7-H4, PD-L1, and ICOS in micro-satellite stable (MSS) and micro-satellite instable (MSI) endometrial tumors

Background: Endometrial cancer is the most frequent tumors of the female reproductive system. While 5-year survival rates are greater than 90% for patients with low histological grade endometrial tumors, survival rates drop for patients with high grade endometrial tumors. With the recent success in checkpoint targeting therapies, particularly in genomic instable tumors, we wanted to characterize the immune infiltrate in a cohort of endometrial tumors. B7-H4 and PD-L1 are known members of checkpoint inhibitory pathways that regulate T cell activity, and have been shown to play a role in promoting immunogenic tolerance in tumors. Both these receptors have been shown to be expressed by tumor cells and tumor-associated macrophages (TAM). ICOS (Inducible CO-Stimulator molecule), a member of the CD28 superfamily expressed primarily on T lymphocytes, has been identified as a potentially key molecule in providing optimal anti-tumor benefit following anti-CTLA-4 therapy. Preclinical data have confirmed that engagement of the ICOS pathway plays a crucial role in mediating anti-tumor responses to checkpoint inhibitors. Published reports have shown that PD-L1 expression is strongly associated with MSI status and can enrich for response to anti-PD-1 therapy. To understand the role of these molecules in mediating immune tolerance we profiled the expression of B7-H4, PD-L1, and ICOS in a cohort of low grade and high grade endometrial patients and correlated with MSI and MSS status. Methods: FFPE tissues from a cohort of 92 patients with endometrial cancer, collected at the Massachusetts General Hospital, were analyzed for expression of B7-H4, PD-L1, and ICOS using established immunohistochemistry (IHC) protocols. Microsatellite instability status was assessed for approximately 50 tumors using an IHC assay for expression of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2. Additionally, ICOS expression on intratumoral T cells was evaluated using RNA sequencing data collected from Cancer Genome Atlas (TCGA). Results: Staining of endometrial cancer samples showed that while a majority of endometrial tumors expressed B7-H4, only a small subset expressed PD-L1. No significant overlap was observed in tumors expressing both inhibitory ligands suggesting that these mechanisms are mutually exclusive in the generation of immune tolerance process. The MSI status was determined by evaluating the expression of miss-match repair genes (MMR) by IHC as a surrogate for genomic stability. We observed that PD-L1 expression was strongly associated with microsatellite instable (MSI) tumors (∼40%) with little-to-no expression of PD-L1 observed in the microsatellite stable (MSS) patients. In both sub-sets, PD-L1 expression was observed predominantly on the infiltrating tumor cells. In contrast, B7-H4 was observed to be in both MSI and MSS sub-set of tumors, however higher levels of B7-H4 (2+/3+) were observed in MSI compared to the MSS subset. In addition to inhibitory ligands we evaluated expression of ICOS on the immune infiltrates. High infiltration of ICOS + cells was observed primarily in the MSI subset. This is consistent with published literature showing high numbers of T cells are associated with the MSI phenotype. Similar data was observed with the POLE mutant tumors that also have high levels of mutations. Together, these data support development and use of B7-H4 or ICOS targeted therapeutics for treatment of MSI sub-set of endometrial tumors. Citation Format: Amit Deshpande, Whitfield B. Growdon, Heather Hirsch, Tong Zi, Chris Grange, Jason Reeves, Jennifer Stall, Bo Rueda, Sriram Sathyanarayanan. Characterization of immune regulatory molecules B7-H4, PD-L1, and ICOS in micro-satellite stable (MSS) and micro-satellite instable (MSI) endometrial tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A052.