Discovery of 1'-(1-phenylcyclopropane-carbonyl)-3H-spiro[isobenzofuran-1,3'-pyrrolidin]-3-one as a novel steroid mimetic scaffold for the potent and tissue-specific inhibition of 11ß-HSD1 using a scaffold-hopping approach.

11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) has been identified as the primary enzyme responsible for the activation of hepatic cortisone to cortisol in specific peripheral tissues resulting in the concomitant antagonism of insulin action within these tissues. Dysregulation of 11ß-HSD1, particularly in adipose tissues, has been associated with metabolic syndrome and type 2 diabetes mellitus. Therefore, inhibition of 11ß-HSD1 with a small nonsteroidal molecule is therapeutically desirable. Implementation of a scaffold-hopping approach revealed a three-point pharmacophore for 11ß-HSD1 that was utilized to design a steroid mimetic scaffold. Reiterative optimization provided valuable insight into the bioactive conformation of our novel scaffold and led to the discovery of INCB13739. Clinical evaluation of INCB13739 confirmed for the first time that tissue-specific inhibition of 11ß-HSD1 in patients with type 2 diabetes mellitus was efficacious in controlling glucose levels and reducing cardiovascular risk factors.

Phase 1/2 Study of the Pan-PIM Kinase Inhibitor INCB053914 Alone or in Combination With Standard-of-Care Agents in Patients With Advanced Hematologic Malignancies.

BACKGROUND: The Proviral Integration site of Moloney murine leukemia virus (PIM) kinases are implicated in tumorigenesis; the pan-PIM kinase inhibitor, INCB053914, demonstrated antitumor activity in hematologic malignancy preclinical models. PATIENTS AND METHODS: This phase 1/2 study evaluated oral INCB053914 alone or combined with standard-of-care agents for advanced hematologic malignancies (NCT02587598). In Parts 1/2 (monotherapy), patients (≥18 years) had acute leukemia, high-risk myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasm, myelofibrosis (MF), multiple myeloma, or lymphoproliferative neoplasms. In Parts 3/4 (combination therapy), patients had relapsed/refractory or newly diagnosed (≥65 years, unfit for intensive chemotherapy) acute myeloid leukemia (AML) or MF with suboptimal ruxolitinib response. RESULTS: Parts 1/2 (n = 58): 6 patients experienced dose-limiting toxicities (DLTs), most commonly aspartate aminotransferase/alanine aminotransferase-elevated (AST/ALT; each n = 4). Fifty-seven patients (98.3%) had treatment-emergent adverse events (TEAEs), most commonly ALT-elevated and fatigue (36.2% each); 48 (82.8%) had grade ≥3 TEAEs, most commonly anemia (31.0%); 8 (13.8%) had grade ≥3 ALT/AST-elevated TEAEs. Parts 3/4 (n = 39): for INCB053914 + cytarabine (AML; n = 6), 2 patients experienced DLTs (grade 3 maculopapular rash, n = 1; grade 3 ALT-elevated and grade 4 hypophosphatemia, n = 1); for INCB053914 + azacitidine (AML; n = 16), 1 patient experienced a DLT (grade 3 maculopapular rash). Two complete responses were observed (1 with incomplete count recovery). For INCB053914 + ruxolitinib (MF; n = 17), no DLTs occurred; 3 patients achieved best reduction of >25% spleen volume at week 12 or 24. CONCLUSION: INCB053914 was generally well tolerated as monotherapy and in combinations; TEAEs were most commonly ALT/AST-elevated. Limited responses were observed with combinations. Future studies are needed to identify rational, effective combination strategies.

Characterization of INCB086550: A Potent and Novel Small-Molecule PD-L1 Inhibitor.

Blocking the activity of the programmed cell death protein 1 (PD-1) inhibitory receptor with therapeutic antibodies against either the ligand (PD-L1) or PD-1 itself has proven to be an effective treatment modality for multiple cancers. Contrasting with antibodies, small molecules could demonstrate increased tissue penetration, distinct pharmacology, and potentially enhanced antitumor activity. Here, we describe the identification and characterization of INCB086550, a novel, oral, small-molecule PD-L1 inhibitor. In vitro, INCB086550 selectively and potently blocked the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, and induced stimulation-dependent cytokine production in primary human immune cells. In vivo, INCB086550 reduced tumor growth in CD34+ humanized mice and induced T-cell activation gene signatures, consistent with PD-L1/PD-1 pathway blockade. Preliminary data from an ongoing phase I study confirmed PD-L1/PD-1 blockade in peripheral blood cells, with increased immune activation and tumor growth control. These data support continued clinical evaluation of INCB086550 as an alternative to antibody-based therapies. SIGNIFICANCE: We have identified a potent small-molecule inhibitor of PD-L1, INCB086550, which has biological properties similar to PD-L1/PD-1 monoclonal antibodies and may represent an alternative to antibody therapy. Preliminary clinical data in patients demonstrated increased immune activation and tumor growth control, which support continued clinical evaluation of this approach. See related commentary by Capparelli and Aplin, p. 1413. This article is highlighted in the In This Issue feature, p. 1397.

Compelling P1 substituent affect on metalloprotease binding profile enables the design of a novel cyclohexyl core scaffold with excellent MMP selectivity and HER-2 sheddase inhibition.

A serendipitous discovery that the metalloprotease binding profile of a novel class of 2-carboxamide-3-hydroxamic acid piperidines could be significantly attenuated by the modification of the unexplored P1 substituent enabled the design and synthesis of a novel 2-carboxamide-1-hydroxamic acid cyclohexyl scaffold core that exhibited excellent HER-2 potency and unprecedented MMP-selectivity that we believe would not have been possible via conventional P1' perturbations.

Conversion of an MMP-potent scaffold to an MMP-selective HER-2 sheddase inhibitor via scaffold hybridization and subtle P1' permutations.

A series of beta-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epidermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, -2, -3, and -9. This was achieved by exploiting subtle differences within the otherwise highly conserved S(1)(') binding pocket of the active sites within the metalloprotease family. In addition, it was discovered that the introduction of polarity to the P(1) and P(1)(') groups reduced the projected human clearance.

Design and identification of selective HER-2 sheddase inhibitors via P1' manipulation and unconventional P2' perturbations to induce a molecular metamorphosis.

In an effort to obtain a MMP selective and potent inhibitor of HER-2 sheddase (ADAM-10), the P1' group of a novel class of (6S,7S)-7-[(hydroxyamino)carbonyl]-6-carboxamide-5-azaspiro[2.5]octane-5-carboxylates was attenuated and the structure-activity relationships (SAR) will be discussed. In addition, it was discovered that unconventional perturbation of the P2' moiety could confer MMP selectivity, which was hypothesized to be a manifestation of the P2' group effecting global conformational changes.

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies.

The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase-signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. In vitro, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated ex vivo and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. In vivo, single-agent INCB053914 inhibited Bcl-2-associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.

Discovery of a potent, selective, and orally active human epidermal growth factor receptor-2 sheddase inhibitor for the treatment of cancer.

The design, synthesis, evaluation, and identification of a novel class of (6S,7S)-N-hydroxy-6-carboxamide-5-azaspiro[2.5]octane-7-carboxamides as the first potent and selective inhibitors of human epidermal growth factor receptor-2 (HER-2) sheddase is described. Several compounds were identified that possess excellent pharmacodynamic and pharmacokinetic properties and were shown to decrease tumor size, cleaved HER-2 extracellular domain plasma levels, and potentiate the effects of the humanized anti-HER-2 monoclonal antibody (trastuzumab) in vivo in a HER-2 overexpressing cancer murine xenograft model.

Analysis of Epstein-Barr virus reservoirs in paired blood and breast cancer primary biopsy specimens by real time PCR.

INTRODUCTION: Epstein-Barr virus (EBV) is present in over 90% of the world's population. This infection is considered benign, even though in limited cases EBV is associated with infectious and neoplastic conditions. Over the past decade, the EBV association with breast cancer has been constantly debated. Adding to this clinical and biological uncertainty, different techniques gave contradictory results for the presence of EBV in breast carcinoma specimens. In this study, minor groove binding (MGB)-TaqMan real time PCR was used to detect the presence of EBV DNA in both peripheral blood and tumor samples of selected patients. METHODS: Peripheral blood and breast carcinoma specimens from 24 patients were collected. DNA was extracted and then amplified by MGB-TaqMan real time PCR. RESULTS: Of 24 breast tumor specimens, 11 (46%) were positive for EBV DNA. Of these 11 breast tumor specimens, 7 (64%) were also positive for EBV DNA in the peripheral blood, while 4 (36%) were positive for EBV DNA in the tumor, but negative in the blood. CONCLUSION: EBV was found at extremely low levels, with a mean of 0.00004 EBV genomes per cell (range 0.00014 to 0.00001 EBV genomes per cell). Furthermore, our finding of the presence of EBV in the tumor specimens coupled to the absence of detection of EBV genomic DNA in the peripheral blood is consistent with the epithelial nature of the virus. Because of the low levels of viral DNA in tumor tissue, further studies are needed to assess the biological input of EBV in breast cancer.

Selective inhibition of ADAM metalloproteases blocks HER-2 extracellular domain (ECD) cleavage and potentiates the anti-tumor effects of trastuzumab.

The HER-2 receptor tyrosine kinase is an important regulator of cell proliferation and survival, and it is a clinically validated target of therapeutic intervention for HER-2 positive breast cancer patients. Its extracellular domain (ECD) is frequently cleaved by protease(s) in HER-2 overexpressing breast cancer patients, rendering the remaining membrane-bound portion (p95) a constitutively activated kinase. The presence of both serum ECD and cellular p95 protein has been linked to poor clinical outcome as well as reduced effectiveness of some therapeutic treatments. We have identified a series of potent, selective small molecule inhibitors of ADAM proteases, exemplified here by INCB003619, and demonstrate that these inhibitors effectively block HER-2 cleavage in HER-2 overexpressing human breast cancer cell lines. Intriguingly, when used in combination, INCB003619 dramatically enhances the antiproliferative activity of suboptimal doses of the anti-HER-2 antibody, trastuzumab, in HER-2 overexpressing/shedding breast cancer cell lines, accompanied by reduced ERK and AKT phosphorylation. Furthermore, INCB003619, in combination with trastuzumab, augments the pro-apoptotic and antiproliferative effects of the chemotherapeutic agent paclitaxel. Consistent with these in vitro data, INCB003619 reduces serum ECD levels and enhances the antitumor effect of trastuzumab in a xenograft tumor model derived from the HER-2 overexpressing BT-474 breast cancer cell line. Collectively, these findings suggest that blocking HER-2 cleavage with selective ADAM inhibitors may represent a novel therapeutic approach for treating HER-2 overexpressing breast cancer patients.

Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells.

Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

A novel kinase inhibitor, INCB28060, blocks c-MET-dependent signaling, neoplastic activities, and cross-talk with EGFR and HER-3.

PURPOSE: The c-MET receptor tyrosine kinase plays important roles in the formation, progression, and dissemination of human cancer and presents an attractive therapeutic target. This study describes the preclinical characterization of INCB28060, a novel inhibitor of c-MET kinase. EXPERIMENTAL DESIGN: Studies were conducted using a series of in vitro and in vivo biochemical and biological experiments. RESULTS: INCB28060 exhibits picomolar enzymatic potency and is highly specific for c-MET with more than 10,000-fold selectivity over a large panel of human kinases. This inhibitor potently blocks c-MET phosphorylation and activation of its key downstream effectors in c-MET-dependent tumor cell lines. As a result, INCB28060 potently inhibits c-MET-dependent tumor cell proliferation and migration and effectively induces apoptosis in vitro. Oral dosing of INCB28060 results in time- and dose-dependent inhibition of c-MET phosphorylation and tumor growth in c-MET-driven mouse tumor models, and the inhibitor is well tolerated at doses that achieve complete tumor inhibition. In a further exploration of potential interactions between c-MET and other signaling pathways, we found that activated c-MET positively regulates the activity of epidermal growth factor receptors (EGFR) and HER-3, as well as expression of their ligands. These effects are reversed with INCB28060 treatment. Finally, we confirmed that circulating hepatocyte growth factor levels are significantly elevated in patients with various cancers. CONCLUSIONS: Activated c-MET has pleiotropic effects on multiple cancer-promoting signaling pathways and may play a critical role in driving tumor cell growth and survival. INCB28060 is a potent and selective c-MET kinase inhibitor that may have therapeutic potential in cancer treatment.

Liver transplantation as definitive treatment for a factor V Leiden mutation.

Liver transplantation (LT) was achieved for factor V Leiden-induced thrombophilia in a neonate with hepatic veno-occlusive disease. Initial LT was performed with a liver segment removed from a child with primary oxalosis. Four months later, a second, definitive LT was performed. The child remains well without recurrent thrombosis.