Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo.

B-cell maturation antigen (BCMA) is an attractive therapeutic target highly expressed on differentiated plasma cells in multiple myeloma and other B-cell malignancies. GSK2857916 (belantamab mafodotin, BLENREP) is a BCMA-targeting antibody-drug conjugate approved for the treatment of relapsed/refractory multiple myeloma. We report that GSK2857916 induces immunogenic cell death in BCMA-expressing cancer cells and promotes dendritic cell activation in vitro and in vivo GSK2857916 treatment enhances intratumor immune cell infiltration and activation, delays tumor growth, and promotes durable complete regressions in immune-competent mice bearing EL4 lymphoma tumors expressing human BCMA (EL4-hBCMA). Responding mice are immune to rechallenge with EL4 parental and EL4-hBCMA cells, suggesting engagement of an adaptive immune response, immunologic memory, and tumor antigen spreading, which are abrogated upon depletion of endogenous CD8+ T cells. Combinations with OX40/OX86, an immune agonist antibody, significantly enhance antitumor activity and increase durable complete responses, providing a strong rationale for clinical evaluation of GSK2857916 combinations with immunotherapies targeting adaptive immune responses, including T-cell-directed checkpoint modulators.

Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines.

The BRAF and MEK Inhibitors Dabrafenib and Trametinib: Effects on Immune Function and in Combination with Immunomodulatory Antibodies Targeting PD-1, PD-L1, and CTLA-4.

PURPOSE: To assess the immunologic effects of dabrafenib and trametinib in vitro and to test whether trametinib potentiates or antagonizes the activity of immunomodulatory antibodies in vivo. EXPERIMENTAL DESIGN: Immune effects of dabrafenib and trametinib were evaluated in human CD4(+) and CD8(+) T cells from healthy volunteers, a panel of human tumor cell lines, and in vivo using a CT26 mouse model. RESULTS: Dabrafenib enhanced pERK expression levels and did not suppress human CD4(+) or CD8(+) T-cell function. Trametinib reduced pERK levels, and resulted in partial/transient inhibition of T-cell proliferation/expression of a cytokine and immunomodulatory gene subset, which is context dependent. Trametinib effects were partially offset by adding dabrafenib. Dabrafenib and trametinib in BRAF V600E/K, and trametinib in BRAF wild-type tumor cells induced apoptosis markers, upregulated HLA molecule expression, and downregulated certain immunosuppressive factors such as PD-L1, IL1, IL8, NT5E, and VEGFA. PD-L1 expression in tumor cells was upregulated after acquiring resistance to BRAF inhibition in vitro. Combinations of trametinib with immunomodulators targeting PD-1, PD-L1, or CTLA-4 in a CT26 model were more efficacious than any single agent. The combination of trametinib with anti-PD-1 increased tumor-infiltrating CD8(+) T cells in CT26 tumors. Concurrent or phased sequential treatment, defined as trametinib lead-in followed by trametinib plus anti-PD-1 antibody, demonstrated superior efficacy compared with anti-PD-1 antibody followed by anti-PD-1 plus trametinib. CONCLUSION: These findings support the potential for synergy between targeted therapies dabrafenib and trametinib and immunomodulatory antibodies. Clinical exploration of such combination regimens is under way.

Centrosomal Chk2 in DNA damage responses and cell cycle progression.

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.

The ubiquitin-proteasome system and assays to determine responses to inhibitors.

IMPORTANCE OF THE FIELD: Proteasome inhibition is an important therapeutic modality. Additionally, given the toxicities of direct proteasome inhibition, interest is increasing in modulating the ubiquitin ligases in the ubiquitin-proteasome system (UPS). AREAS COVERED IN THIS REVIEW: A detailed examination of the ubiquitin-proteasome pathway and an examination of methods of inhibiting this pathway from a variety of targets including the proteasome, the ubiquitin ligases and molecular biology techniques. Special attention is given to the assays used to measure modulation of the ubiquitin-proteasome pathway. WHAT THE READER WILL GAIN: A thorough examination of the UPS and its role in cells and disease and an overview of several assays for analyzing the effect of inhibitors on the UPS. Significant detail is given to assays of the ligase system and molecular approaches. These assays have their own advantages and disadvantages and will allow investigators to make informed choices on investigating the UPS. TAKE HOME MESSAGE: Interrupting the UPS can have profound consequences for cellular health and disease progression. The ubiquitin-proteasome pathway contains multiple activities that cannot be definitively assayed by a single technique. Assaying the UPS requires investigators to use multiple corroborating techniques and avoid confounding issues within each technique.

The Plk1 Polo box domain mediates a cell cycle and DNA damage regulated interaction with Chk2.

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Chk2 is a tumor suppressor that participates in DNA damage checkpoint signaling cascades. Plk1 phosphorylates, colocalizes with, and interacts with Chk2, suggesting interconnection of DNA damage checkpoints and mitotic regulation. However, the function of their association is unknown. Here, we show that the interaction between Chk2 and Plk1 is cell cycle-regulated, with a peak in mitosis. DNA damage in G2 and M phases but not in S phase induces dissociation of Plk1 and Chk2. In vitro, the Plk1 PBD binds phosphorylated Chk2, and mediates an interaction independent of other eukaryotic proteins. Additionally, a phosphopeptide encompassing phosphoT68 of Chk2 binds Plk1 in a PBD-dependent manner, and stimulates Plk1 activity. These results identify potential mechanisms for interaction and inter-regulation of these two protein kinases.

Interaction of chromatin-associated Plk1 and Mcm7.

Plk1 is a multifunctional protein kinase involved in regulation of mitotic entry, chromosome segregation, centrosome maturation, and mitotic exit. Plk1 is a target of DNA damage checkpoints and aids resumption of the cell cycle during recovery from G2 arrest. The polo-box domain (PBD) of Plk1 interacts with phosphoproteins and localizes Plk1 to some mitotic structures. In a search for proteins that interact with the PBD of Plk1, we identified two of the minichromosome maintenance (MCM) proteins, Mcm2 and Mcm7. Co-immunoprecipitation and immunoblot analysis showed an interaction between full-length Plk1 and all other members of the MCM2-7 protein complex. Endogenous Plk1 co-immunoprecipitates with basal forms of Mcm7 as well as with slower migrating forms of Mcm7, induced in response to DNA damage. The strongest interaction between endogenous Plk1 and Mcm7 was detected in a soluble chromatin fraction. These findings suggest a new function for Plk1 in coordination of DNA replication and mitotic events.

Phosphorylation of Plk1 at S137 and T210 is inhibited in response to DNA damage.

Polo-like kinase 1 (Plk1) regulates multiple processes during mitosis. Plk1 is activated by phosphorylation at the G2/M phase boundary. Active Plk1 is involved in promotion of mitotic entry through activation of Cdc25C, and through nuclear import of cyclin B1 that together activate Cdc2/cyclin B kinase. In earlier work, phosphopeptide mapping identified several phosphorylation sites in Plk1. Mutational analysis pinpointed threonine 210, which is located in the activation loop of the kinase domain, as the major activation site of Plk1. In response to DNA damage, ATM/ATR-dependent checkpoint pathways inhibit Plk1 activity. Insensitivity of Plk1T210D, a constitutively active mutant, to DNA damage-induced inhibition of Plk1 indicates that regulation of Plk1 phosphorylation is a potential target of DNA damage checkpoints. In the present paper, we report that in vivo phosphorylation of Plk1 at serine 137 (S137) and threonine 210 (T210) occurs in mitosis. DNA damage prevents phosphorylation of Plk1 at both S137 and T210 in asynchronous cells but not in mitotic cells. Inhibitors of ATM/ATR and Chk1/Chk2 protein kinases avert the inhibition of Plk1 phosphorylation in response to DNA damage. These data suggest a participation of DNA damage checkpoints in regulation of the signaling pathways upstream of Plk1.

Polo-like kinases and Chk2 at the interface of DNA damage checkpoint pathways and mitotic regulation.

The cell cycle controls processes of DNA replication and segregation of replicated DNA into two daughter cells. These processes are coordinated by multiple signaling pathways, which employ many protein kinases. The members of the family of Polo-like protein kinases are among these key cell cycle regulators. In response to DNA damage and inhibited DNA replication, DNA structure checkpoints delay cell cycle progression to provide cells with time for repair of damaged DNA and protect it from more severe damage. These effects are achieved by affecting key players of the basic cell cycle regulation of the cells with damaged DNA. This review is focused on the interplay between Chk2, a bona fide checkpoint protein kinase, and Polo-like kinases.

Polo-like kinase 1 and Chk2 interact and co-localize to centrosomes and the midbody.

Chk2 is a protein kinase intermediary in DNA damage checkpoint pathways. DNA damage induces phosphorylation of Chk2 at multiple sites concomitant with activation. Chk2 phosphorylated at Thr-68 is found in nuclear foci at sites of DNA damage (1). We report here that Chk2 phosphorylated at Thr-68 and Thr-26 or Ser-28 is localized to centrosomes and midbodies in the absence of DNA damage. In a search for interactions between Chk2 and proteins with similar subcellular localization patterns, we found that Chk2 coimmunoprecipitates with Polo-like kinase 1, a regulator of chromosome segregation, mitotic entry, and mitotic exit. Plk1 overexpression enhances phosphorylation of Chk2 at Thr-68. Plk1 phosphorylates recombinant Chk2 in vitro. Indirect immunofluorescence (IF) microscopy revealed the co-localization of Chk2 and Plk1 to centrosomes in early mitosis and to the midbody in late mitosis. These findings suggest lateral communication between the DNA damage and mitotic checkpoints.

Chk2 activation and phosphorylation-dependent oligomerization.

The tumor suppressor gene CHK2 encodes a versatile effector serine/threonine kinase involved in responses to DNA damage. Chk2 has an amino-terminal SQ/TQ cluster domain (SCD), followed by a forkhead-associated (FHA) domain and a carboxyl-terminal kinase catalytic domain. Mutations in the SCD or FHA domain impair Chk2 checkpoint function. We show here that autophosphorylation of Chk2 produced in a cell-free system requires trans phosphorylation by a wortmannin-sensitive kinase, probably ATM or ATR. Both SQ/TQ sites and non-SQ/TQ sites within the Chk2 SCD can be phosphorylated by active Chk2. Amino acid substitutions in the SCD and the FHA domain impair auto- and trans-kinase activities of Chk2. Chk2 forms oligomers that minimally require the FHA domain of one Chk2 molecule and the SCD within another Chk2 molecule. Chk2 oligomerization in vivo increases after DNA damage, and when damage is induced by gamma irradiation, this increase requires ATM. Chk2 oligomerization is phosphorylation dependent and can occur in the absence of other eukaryotic proteins. Chk2 can cross-phosphorylate another Chk2 molecule in an oligomeric complex. Induced oligomerization of a Chk2 chimera in vivo concomitant with limited DNA damage augments Chk2 kinase activity. These results suggest that Chk2 oligomerization regulates Chk2 activation, signal amplification, and transduction in DNA damage checkpoint pathways.