INDUCE-2: A Phase I/II, open-label, two-part study of feladilimab in combination with tremelimumab in patients with advanced solid tumors.

Combining immunotherapies with distinct mechanisms of action has the potential to overcome treatment resistance and improve outcomes. The inducible T-cell co-stimulator (ICOS) agonist feladilimab is directed at enhancing T-cell activation and function, thereby promoting an antitumor response. INDUCE-2 (NCT03693612) was a Phase I/II, open-label, two-part study evaluating the anti-ICOS agonist feladilimab in combination with the anti-CTLA-4 antibody tremelimumab in patients with select advanced solid tumors. Objectives of Part 1 were to determine the safety, tolerability, and recommended phase 2 dose (RP2D) of feladilimab in combination with tremelimumab. In Part 2, the antitumor activity of the combination (administered at the RP2D determined in Part 1) was to be assessed in patients with relapsed/refractory head and neck squamous cell carcinoma. Primary endpoints included the rates of dose-limiting toxicities (DLTs), adverse events (AEs), AEs of special interest, and serious AEs. Secondary endpoints included overall response rate, while biomarker assessment was exploratory. A total of 26 patients were enrolled, 18 (69%) of whom had completed the study at end date. One patient, in the highest dose group (24/225 mg feladilimab/tremelimumab), experienced a DLT 18 days after the first dose of study treatment. All patients experienced at least one AE; AEs led to treatment discontinuation in four (15%) patients. Partial response was observed in one patient. Feladilimab in combination with tremelimumab was well-tolerated but showed limited efficacy. Based on the totality of data from Part 1, it was decided not to continue with Part 2.

Gap junction coupling is required for tumor cell migration through lymphatic endothelium.

OBJECTIVE: The lymphatic vasculature is a well-established conduit for metastasis, but the mechanisms by which tumor cells interact with lymphatic endothelial cells (LECs) to facilitate escape remain poorly understood. Elevated levels of the lymphangiogenic peptide adrenomedullin are found in many tumors, and we previously characterized that its expression is necessary for lymphatic vessel growth within both tumors and sentinel lymph nodes and for distant metastasis. APPROACH AND RESULTS: This study used a tumor cell-LEC coculture system to identify a series of adrenomedullin-induced events that facilitated transendothelial migration of the tumor cells through a lymphatic monolayer. High levels of adrenomedullin expression enhanced adhesion of tumor cells to LECs, and further analysis revealed that adrenomedullin promoted gap junction coupling between LECs as evidenced by spread of Lucifer yellow dye. Adrenomedullin also enhanced heterocellular gap junction coupling as demonstrated by Calcein dye transfer from tumor cells into LECs. This connexin-mediated gap junction intercellular communication was necessary for tumor cells to undergo transendothelial migration because pharmacological blockade of this heterocellular communication prevented the ability of tumor cells to transmigrate through the lymphatic monolayer. In addition, treatment of LECs with adrenomedullin caused nuclear translocation of ß-catenin, a component of endothelial cell junctions, causing an increase in transcription of the downstream target gene C-MYC. Importantly, blockade of gap junction intercellular communication prevented ß-catenin nuclear translocation. CONCLUSIONS: Our findings indicate that maintenance of cell-cell communication is necessary to facilitate a cascade of events that lead to tumor cell migration through the lymphatic endothelium.

Adrenomedullin gene dosage correlates with tumor and lymph node lymphangiogenesis.

Adrenomedullin (AM) is a potent lymphangiogenic factor that promotes lymphatic endothelial cell (LEC) proliferation through a pharmacologically tractable G-protein-coupled receptor. Numerous types of human cancers have increased levels of AM; however, the functional consequences of this fact have not been characterized. Therefore, we evaluated whether modulating adrenomedullin (Adm) gene dosage within tumor cells affects lymphangiogenesis. Murine Lewis lung carcinoma (LLC) cells that overexpress or underexpress Adm were injected subcutaneously into C57BL/6 mice, and tumors were evaluated for growth and vascularization. A dosage range from ∼10 to 200% of wild-type Adm expression did not affect LLC proliferation in vitro or in vivo, nor did it affect angiogenesis. Notably, the dosage of Adm markedly and significantly influenced tumor lymphangiogenesis. Reduced Adm expression in tumors decreased the proliferation of LECs and the number of lymphatic vessels, while elevated tumor Adm expression led to enlarged lymphatic vessels. Moreover, overexpression of Adm in tumors induced sentinel lymph node lymphangiogenesis and led to an increased incidence of Ki67-positive foci within the lung. These data show that tumor-secreted AM is a critical factor for driving both tumor and lymph node lymphangiogenesis. Thus, pharmacological targeting of AM signaling may provide a new avenue for inhibition of tumor lymphangiogenesis.

A Phase I/II Open-Label Study of Molibresib for the Treatment of Relapsed/Refractory Hematologic Malignancies.

PURPOSE: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). PATIENTS AND METHODS: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non-Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). RESULTS: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8-18.7], 25% (95% CI, 7.3-52.4), and 13% (95% CI, 6.9-20.6), respectively. CONCLUSIONS: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.

ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells.

Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death.

Detailing the role of Bax translocation, cytochrome c release, and perinuclear clustering of the mitochondria in the killing of HeLa cells by TNF.

Induction of cell death in HeLa cells with TNF and cycloheximide (CHX) required an adequate ATP supply and was accompanied by decrease in intracellular pH, translocation of Bax, perinuclear clustering of the mitochondria, and cytochrome c release. The chloride channel inhibitor furosemide prevented the intracellular acidification, the translocation of Bax and the cell death. Cyclosporin A (CyA) or bongkrekic acid (BK) inhibited the induction of the MPT, the release of cytochrome c and the cell death without affecting the perinuclear clustering of the mitochondria or the translocation of Bax. Energy depletion with the ATP synthase inhibitor oligomycin or the uncoupler FCCP in the presence of 2-deoxy-glucose prevented the perinuclear clustering of the mitochondria and the cell killing. However, mitochondrial translocation of Bax was still observed. By contrast, cytochrome c was released in the oligomycin-treated cells but not in the same cells treated with FCCP. The data demonstrate that apoptosis in HeLa cells is ATP dependent and requires the translocation of Bax. The movement of Bax to the mitochondria occurs before and during the perinuclear clustering of these organelles and does not require the presence of ATP. The release of cytochrome c depends on the induction of the mitochondrial permeability transition but not ATP content.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers.

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

Schlemm's canal: more than meets the eye, lymphatics in disguise.

Schlemm's canal (SC) is a unique vascular structure that functions to maintain fluid homeostasis by draining aqueous humor from the eye into the systemic circulation. The endothelium lining the inner wall of SC has both blood and lymphatic vascular characteristics, thus prompting exploration of the development and regulation of this unique channel. In this issue of the JCI, back-to-back papers by Aspelund et al. and Park et al. detail the mechanisms of SC development, which includes a lymphatic reprogramming that is necessary to maintain proper function. Furthermore, both groups exploit the lymph-like qualities of this canal: they identify VEGF-C as a potential therapeutic for glaucoma and suggest that expression of PROX1, a marker of lymphatic fate, could also serve as a biosensor for monitoring SC integrity. These studies provide substantial insight into the molecular and cellular pathways that govern SC development and reveal that ocular pathology is associated with deregulation of the lymph-like characteristics of SC.

Decoy receptor CXCR7 modulates adrenomedullin-mediated cardiac and lymphatic vascular development.

Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm, gene; AM, protein)-a mitogenic peptide hormone required for normal cardiovascular development-is tightly controlled by CXCR7. To this end, Cxcr7(-/-) mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes that can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7(-/-) mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development.

Cooperativity between MAPK and PI3K signaling activation is required for glioblastoma pathogenesis.

BACKGROUND: Glioblastoma (GBM) genomes feature recurrent genetic alterations that dysregulate core intracellular signaling pathways, including the G1/S cell cycle checkpoint and the MAPK and PI3K effector arms of receptor tyrosine kinase (RTK) signaling. Elucidation of the phenotypic consequences of activated RTK effectors is required for the design of effective therapeutic and diagnostic strategies. METHODS: Genetically defined, G1/S checkpoint-defective cortical murine astrocytes with constitutively active Kras and/or Pten deletion mutations were used to systematically investigate the individual and combined roles of these 2 RTK signaling effectors in phenotypic hallmarks of glioblastoma pathogenesis, including growth, migration, and invasion in vitro. A novel syngeneic orthotopic allograft model system was used to examine in vivo tumorigenesis. RESULTS: Constitutively active Kras and/or Pten deletion mutations activated both MAPK and PI3K signaling. Their combination led to maximal growth, migration, and invasion of G1/S-defective astrocytes in vitro and produced progenitor-like transcriptomal profiles that mimic human proneural GBM. Activation of both RTK effector arms was required for in vivo tumorigenesis and produced highly invasive, proneural-like GBM. CONCLUSIONS: These results suggest that cortical astrocytes can be transformed into GBM and that combined dysregulation of MAPK and PI3K signaling revert G1/S-defective astrocytes to a primitive gene expression state. This genetically-defined, immunocompetent model of proneural GBM will be useful for preclinical development of MAPK/PI3K-targeted, subtype-specific therapies.

Adrenomedullin Function in Vascular Endothelial Cells: Insights from Genetic Mouse Models.

Adrenomedullin is a highly conserved peptide implicated in a variety of physiological processes ranging from pregnancy and embryonic development to tumor progression. This review highlights past and present studies that have contributed to our current appreciation of the important roles adrenomedullin plays in both normal and disease conditions. We provide a particular emphasis on the functions of adrenomedullin in vascular endothelial cells and how experimental approaches in genetic mouse models have helped to drive the field forward.

The course of etoposide-induced apoptosis in Jurkat cells lacking p53 and Bax.

Jurkat T-lymphocytes lack p53 and Bax but contain p73 and Bid and are killed by etoposide (ETO). With ETO c-abl is phosphorylated and phosphorylated p73 increased. Translocation of full-length Bid to mitochondria follows, with induction of the mitochondrial permeability transition (MPT) and release of cytochrome c into the cytosol. Pronounced swelling of mitochondria was evident ultrastructurally, and the MPT inhibitor cyclosporin A prevented the release of cytochrome c. Overexpression of Bcl-2 prevented the translocation of Bid, the release of cytochrome c, and cell death. The pan-caspase inhibitor ZVAD-FMK prevented the cell killing, but not the initial release of cytochrome c. An accumulation of tBid occurred at later times in association with Bid degradation. A sequence is proposed that couples DNA damage to Bid translocation via activation of c-abl and p73. Bid translocation induces the MPT, the event that causes release of cytochrome c, activation of caspases, and cell death.

Re-evaluation of the distinction between type I and type II cells: the necessary role of the mitochondria in both the extrinsic and intrinsic signaling pathways upon Fas receptor activation.

Cyclosporin A (CyA) and bongkrekic acid (BK) prevented Fas-induced apoptosis in two type I cell lines (H9 and SKW6.4) and two type II cell lines (Jurkat and CEM). CyA and BK inhibited the release of cytochrome c in all four cell lines. In type I cells and in CEM cells, CyA and BK did not prevent the translocation of Bax to the mitochondria. In these same cells, full-length Bid decreased in the mitochondria and cytosol. The cleavage product of Bid, tBid, appeared in the cytosol and to a lesser extent in the mitochondria. In Jurkat cells, Bid also decreased in the cytosol, but increased in the mitochondria. Similar to the other cells, tBid appeared in the mitochondria and cytosol. In the type I H9 and SKW6.4 cells and type II Jurkat cells, the caspase-8 inhibitor Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (IETD) prevented the cell killing. In the type I cells, IETD prevented the translocation of Bax, the degradation of Bid and the accumulation of tBid. By contrast, IETD only marginally protected the type II CEM cells. In these cells in the presence of IETD, Bax translocated to the mitochondria, in the absence of any degradation of Bid or accumulation of tBid. In the type I H9 cells, IETD produced a depletion of ATP, an effect that did not occur in the type II CEM cells. It is concluded that in type I cells the extrinsic signaling pathway is mitochondrial dependent to the same extent as is the intrinsic pathway in type II cells.

The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c.

Treatment of L929 fibroblasts by the topoisomerase II inhibitor etoposide killed 50% of the cells within 72 h. The cell killing was preceded by the release of cytochrome c from the mitochondria. Simultaneous treatment of the cells with wortmannin, cycloheximide, furosemide, cyclosporin A, or decylubiquinone prevented the release of cytochrome c and significantly reduced the loss of viability. Etoposide caused the phosphorylation of p53 within 6 h, an effect prevented by wortmannin, an inhibitor of DNA-dependent protein kinase (DNA-PK). The activation of p53 by etoposide resulted in the up-regulation of the pro-apoptotic protein Bax, a result that was prevented by the protein synthesis inhibitor cycloheximide. The increase in the content of Bax was followed by the translocation of this protein from the cytosol to the mitochondria, an event that was inhibited by furosemide, a chloride channel inhibitor. Stably transfected L929 fibroblasts that overexpress Akt were resistant to etoposide and did not translocate Bax to the mitochondria or release cytochrome c. Bax levels in these transfected cells were comparable with the wild-type cells. The release of cytochrome c upon translocation of Bax has been attributed to induction of the mitochondrial permeability transition (MPT). Cyclosporin A and decylubiquinone, inhibitors of MPT, prevented the release of cytochrome c without affecting Bax translocation. These data define a sequence of biochemical events that mediates the apoptosis induced by etoposide. This cascade proceeds by coupling DNA damage to p53 phosphorylation through the action of DNA-PK. The activation of p53 increases Bax synthesis. The translocation of Bax to the mitochondria induces the MPT, the event that releases cytochrome c and culminates in the death of the cells.

Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha.

Induction of apoptosis in HeLa cells with staurosporine produced a rise in the intracellular pH (pH(i)). Intracellular alkalinization was accompanied by translocation of Bax to the mitochondria, cytochrome c release, and cell death. The chloride channel inhibitor furosemide prevented intracellular alkalinization, Bax translocation, cytochrome c release, and cell death. Translocation of full-length Bid to the mitochondria was also prevented by furosemide. The cleavage product of Bid degradation (truncated Bid, tBid) was not detectable in the mitochondria. Its accumulation in the cytosol was prevented by furosemide. Apoptosis induced by tumor necrosis factor-alpha (TNF) lowered pH(i), an effect also accompanied by Bax translocation, cytochrome c release, and cell killing. Furosemide prevented all of these events. TNF induced a depletion of full-length Bid from the mitochondria and the cytosol but induced an accumulation of mitochondrial tBid. Furosemide only delayed full-length Bid depletion and tBid accumulation. The caspase 8 inhibitor IETD did not prevent the translocation of Bax. Although IETD did inhibit the cleavage of Bid and the accumulation of tBid, cell killing was reduced only slightly. It is concluded that with either staurosporine or TNF a furosemide-sensitive change in pH(i) is linked to Bax translocation, cytochrome c release, and cell killing. With TNF Bax translocation occurs as Bid is depleted and can be dissociated from the accumulation of tBid. With staurosporine a role for full-length Bid in Bax translocation cannot be excluded but is not necessary as evidenced by the data with TNF.

Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition.

In Jurkat cells Bid was cleaved upon activation of the Fas receptor with an anti-Fas antibody. The caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-CH(2)F (IETD) prevented the cleavage of Bid and the loss of viability. The nuclear enzyme poly(ADP-ribose)polymerase (PARP) was also cleaved upon the activation of caspases, and IETD similarly prevented PARP cleavage. The PARP inhibitor 3-aminobenzamide (3-AB) restored the cell killing in the presence of IETD, an effect that occurred without restoration of the cleavage of Bid or PARP. In the presence of 3-AB and IETD, translocation occurred of full-length Bid to the mitochondria. The induction of the mitochondrial permeability transition (MPT) was documented by the cyclosporin A (CyA) sensitivity of the release of cytochrome c, the release of malate dehydrogenase from the mitochondrial matrix, the loss of the mitochondrial membrane potential, and the pronounced swelling of these organelles, as assessed by electron microscopy. In addition to preventing all evidence of the MPT, CyA prevented the loss of cell viability, without effect on the cleavage of either Bid or PARP. The prevention of PARP cleavage by inhibition of caspase-3 resulted in a 10-fold activation of the enzyme and a resultant depletion of NAD and ATP. The PARP inhibitor 3-AB prevented the loss of NAD and ATP. Depletion of ATP by metabolic inhibitors similarly prevented the cell killing. It is concluded that the cleaving of PARP in Fas-mediated apoptosis allowed expression of an energy-dependent cell death program that included the translocation of full-length Bid to the mitochondria with induction of the MPT.