Design and synthesis of 4th generation EGFR inhibitors against human triple (Del19/T790M/C797S) mutation.

Epidermal growth factor receptor (EGFR)-targeted therapy is used to treat EGFR mutation-induced non-small cell lung cancer (NSCLC). However, its efficacy does not last beyond a certain period due to the development of primary and secondary resistance. First and second-generation inhibitors (e.g., gefitinib, erlotinib, and afatinib) induce EGFR T790M mutations, while third-generation inhibitors (e.g., osimertinib) induce C797S as a major target resistance mutation. Therefore, the C797S mutation is being actively researched. In this study, we investigated the structure-activity relationship of several synthesized compounds as fourth-generation inhibitors against the C797S mutation. We identified a compound 13k that displayed nanomolar potency and high selectivity. Moreover, we used a triple mutant xenograft mouse model to evaluate the in vivo efficacy of 13k in inhibiting EGFR C797S, which demonstrated exceptional profiles and satisfactory EGFR C797S inhibition efficacy. Based on its excellent in vitro and in vivo profiles, compound 13k can be considered a promising candidate for treating EGFR C797S mutations.

Targeting HIF-1α/NOTCH1 pathway eliminates CD44+ cancer stem-like cell phenotypes, malignancy, and resistance to therapy in head and neck squamous cell carcinoma.

Poor prognosis of head and neck squamous cell carcinomas (HNSCCs) results from resistance to chemotherapy and radiotherapy. To uncover the drivers of HNSCC resistance, including stemness and hypoxia, in this study, we compared the gene expression between CD44+ and CD44- HNSCC cells and assessed the correlation of CD44 and hypoxia-inducible factor 1α (HIF-1α) expression with mouse features and outcomes of patients with HNSCC. We combined the knockdown or activation of HIF-1α with in vitro and in vivo assays to evaluate effects on stemness and resistance of HNSCC cells. Analysis of clinical data showed that activation of HIF-1α in CD44+ patients with HNSCC was correlated with worse prognosis. Functional assays showed that HIF-1α promoted stemness, resistance, and epithelial-mesenchymal transition in HNSCC CD44+ cells. HIF-1α activated NOTCH1 signaling in HNSCC stem-like cells characterized by CD44 expression. Moreover, inhibition of these signaling proteins using shRNA or Evofosfamide (Evo) development for cancer treatment, reversed chemoresistance in vitro and in vivo. Taken together, our results indicated that targeting HIF-1α attenuated NOTCH1-induced stemness, which regulates responses to chemotherapy or radiotherapy and malignancy in CD44+ HNSCCs. HIF-1α/NOTCH1 signaling may represent a target for HNSCC treatment.

Triterpenoid pristimerin synergizes with taxol to induce cervical cancer cell death through reactive oxygen species-mediated mitochondrial dysfunction.

A combined treatment with conventional chemotherapies can enhance the effectiveness of chemotherapeutic agents against cancers. Here, we have shown that the naturally occurring triterpenoids synergistically enhance the response of cervical cancer cells to taxol. Of the triterpenoid compounds, pristimerin enhanced the anticancer effect of taxol with the highest efficiency by combination. Pristimerin synergizes with taxol to inhibit clonogenic survival and tumor growth in nude mice, and to enhance cell death in cervical cancer cells. A combined treatment with taxol and pristimerin induced cervical cancer cell death by increasing intracellular reactive oxygen species levels, upregulation of death receptor death receptor 5 (DR5), activation of Bax, and dissipation of mitochondrial membrane potential. Treatment with N-acetyl-L-cysteine, a thiol-containing antioxidant completely blocked combined treatment-induced Bax translocation as well as DR5 upregulation. Moreover, inhibition of Jun N-terminal kinase/c-Jun pathway attenuated cell death by blocking DR5 upregulation and Bax activation. These results indicate that the triterpenoid, pristimerin, synergistically enhances taxol response of cervical cancer cells through DR5 expression and Bax activation. Furthermore, the reactive oxygen species-dependent activation of the Jun N-terminal kinase/c-Jun pathway is required for the DR5 upregulation and Bax activation. The molecular mechanism revealed by this study may aid in the design of future combination cancer therapies against cells with intrinsically reduced sensitivity to taxol.

Target modulation and pharmacokinetics/pharmacodynamics translation of the BTK inhibitor poseltinib for model-informed phase II dose selection.

The selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al. Arthritis Res. Ther. 18, 91, https://doi.org/10.1186/s13075-016-0988-z , 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478].

Activation of p38 mitogen-activated protein kinase is required for death receptor-independent caspase-8 activation and cell death in response to sphingosine.

Sphingosine induces activation of multiple signaling pathways that play critical roles in controlling cell death. However, the precise molecular mechanism of cell death induced by sphingosine remains to be clarified. In this study, we show that sphingosine induces death receptor-independent caspase-8 activation and apoptotic cell death via p38 mitogen-activated protein kinase (MAPK) activation and that suppression of the MAPK/extracellular signal-regulated kinase (ERK) kinase/ERK pathway by protein phosphatase 2A (PP2A) is required for p38 MAPK activation. Treatment of cells with sphingosine induced suppression of ERK and activation of p38 MAPK. Inhibition of p38 MAPK led to the marked suppression of death receptor-independent caspase-8 activation and subsequent cell death induced by sphingosine. Interestingly, pretreatment with phorbol 12-myristate 13-acetate or transfection of MAPK/ERK kinase/ERK resulting in ERK activation completely attenuated sphingosine-induced p38 MAPK activation. PP2A activity was additionally elevated on sphingosine treatment. Small interfering RNA targeting of PP2A effectively attenuated sphingosine-induced p38 MAPK activation through restoration of ERK activity, suggesting PP2A-mediated opposing regulation of ERK and p38 MAPK. Our findings clearly imply that activation of p38 MAPK promotes death receptor-independent activation of caspase-8 and apoptotic cell death pathways, thus providing a novel cellular mechanism for the anticancer activity of sphingolipid metabolites.

Reactive oxygen species-dependent activation of Bax and poly(ADP-ribose) polymerase-1 is required for mitochondrial cell death induced by triterpenoid pristimerin in human cervical cancer cells.

Naturally occurring triterpenoid compounds have long been used as anti-inflammatory, antimalarial, and insecticidal agents. It has become evident that some of the natural or synthetic triterpenoids have promising clinical potential as both a therapeutic and chemopreventive agent for cancer. However, the molecular basis for the antitumor activity of triterpenoid has yet to be defined. In this study, we show that pristimerin, a natural triterpenoid, induces mitochondrial cell death in human cervical cancer cells and that reactive oxygen species (ROS)-dependent activation of both Bax and poly(ADP-ribose) polymerase-1 (PARP-1) is critically required for the mitochondrial dysfunction. We also showed that c-Jun N-terminal kinase (JNK) is involved in ROS-dependent Bax activation. Treatment of pristimerin induced an increase in intracellular ROS, JNK activation, conformational change, and mitochondrial redistribution of Bax, mitochondrial membrane potential loss, and cell death. The PARP-1 was also found to be activated by pristimerin treatment. An antioxidant, N-acetyl-l-cysteine (NAC), inhibited pristimerin-induced JNK activation, Bax relocalization, and PARP-1 activation, as well as mitochondrial cell death. Moreover, inhibition of JNK clearly suppressed conformational change and mitochondrial translocation of Bax and subsequent mitochondrial cell death but did not affect PARP-1 activation. Inhibition of PARP-1 with 1,5-dihydroxyisoquinoline (DIQ) or with small interfering RNA of PARP-1 significantly attenuated pristimerin-induced mitochondrial membrane potential loss and cell death but did not affect JNK activation and Bax relocalization. These results indicate that the natural triterpenoid pristimerin induces mitochondrial cell death through ROS-dependent activation of both Bax and PARP-1 in human cervical cancer cells and that JNK is involved in ROS-dependent Bax activation.

The Rac1/MKK7/JNK pathway signals upregulation of Atg5 and subsequent autophagic cell death in response to oncogenic Ras.

To prevent the development of malignancies, mammalian cells activate disposal programs, such as programmed cell death, in response to deregulated oncogene expression. However, the molecular basis for regulation of cellular disposal machinery in response to activated oncogenes is unclear at present. In this study, we show that upregulation of the autophagy-related protein, Atg5, is critically required for the oncogenic H-ras-induced autophagic cell death and that Rac1/mitogen-activated kinase kinase (MKK) 7/c-Jun N-terminal kinase (JNK) signals upregulation of Atg5. Overexpression of H-ras(V12) induced marked autophagic vacuole formation and cell death in normal fibroblasts, which remained unaffected by a caspase inhibitor. Pretreatment with Bafilomycin A1, an autophagy inhibitor, completely attenuated H-ras(V12)-induced cell death as well as autophagic vacuole formation. Selective production of Atg5 was observed in cells overexpressing H-ras(V12), and small interfering RNA (siRNA) targeting of Atg5 clearly inhibited autophagic cell death. Interestingly, inhibition of JNK or c-Jun by specific siRNA suppressed Atg5 upregulation and autophagic cell death. Moreover, inhibition of MKK7, but not MKK4, effectively attenuated H-ras(V12)-induced JNK activation. In addition, ectopic expression of RacN17 or Rac1-siRNA effectively inhibited MKK7-JNK activation, Atg5 upregulation and autophagic cell death. These data support the notion that upregulation of Atg5 is required for the oncogenic H-ras-induced autophagic cell death in normal fibroblasts and that activation of Rac1/MKK7/JNK-signaling pathway leads to upregulation of Atg5 in response to oncogenic H-ras. Our findings suggest that in cells acquiring deregulated oncogene expression, oncogenic stress triggers autophagic cell death, which protects cells against malignant progression.

c-Src-p38 mitogen-activated protein kinase signaling is required for Akt activation in response to ionizing radiation.

The Akt and mitogen-activated protein kinase (MAPK) pathways have been implicated in tumor cell survival and contribute to radiation resistance. However, the molecular basis for link between MAPK and Akt in cell survival response to radiation is unclear. Here, we show that c-Src-Rac1-p38 MAPK pathway signals Akt activation and cell survival in response to radiation. Ionizing radiation triggered Thr(308) and Ser(473) phosphorylation of Akt. Exposure of cells to radiation also induced p38 MAPK and c-Jun NH(2)-terminal kinase activations. Inhibition of c-Jun NH(2)-terminal kinase suppressed radiation-induced cell death, whereas inhibition of p38 MAPK effectively increased sensitivity to radiation. Interestingly, inhibition of p38 MAPK completely attenuated radiation-induced Ser(473) phosphorylation of Akt but did not affect Thr(308) phosphorylation. Conversely, overexpression of p38 MAPK enhanced Ser(473) phosphorylation of Akt in response to radiation. In addition, inhibition of p38 MAPK failed to alter phosphoinositide 3-kinase and phosphoinositide-dependent protein kinase activities. Ectopic expression of RacN17, dominant-negative form of Rac1, inhibited p38 MAPK activation and Ser(473) phosphorylation of Akt. Following exposure to radiation, c-Src was selectively activated among Src family tyrosine kinases. Inhibition of c-Src attenuated Rac1 and p38 MAPK activations and Ser(473) phosphorylation of Akt. Our results support the notion that the c-Src-Rac1-p38 MAPK pathway is required for activation of Akt in response to radiation and plays a cytoprotective role against radiation in human cancer cells.

Activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death.

Despite extensive investigation, the molecular mechanism of anticancer activity of sphingolipid metabolites remains to be clarified. Here we demonstrate that sphingosine induces mitochondrial cell death via Lck-mediated conformational activation of Bak in Jurkat T cell lymphoma. Treatment of cells with sphingosine rapidly induced mitochondrial membrane potential loss, cytochrome c release from mitochondria, and apoptotic cell death. Sphingosine also induced conformational activation of Bak, but not Bax. siRNA targeting of Bak effectively attenuated sphingosine-induced mitochondrial cell death, indicating that Bak is involved in sphingosine-induced mitochondrial cell death. Sphingosine also induced activation of tyrosine kinase Lck. Inhibition of Lck by treatment of PP2, a Lck inhibitor or siRNA targeting of Lck suppressed sphingosine-induced conformational activation and oligomerization of Bak, mitochondrial membrane potential loss, and apoptotic cell death, implying that activation of Lck is critically required for sphingosine-induced conformational activation of Bak and mitochondrial cell death. The results elucidated in this study provide a novel cellular mechanism for the anticancer activity of sphingolipid metabolites.

HM71224, a selective Bruton's tyrosine kinase inhibitor, attenuates the development of murine lupus.

BACKGROUND: Systemic lupus erythematosus (SLE) is associated with B cell hyperactivity, and lupus nephritis (LN), in particular, is promoted by the production of autoantibodies and immune complex deposition. Bruton's tyrosine kinase (BTK) plays critical roles in B cell receptor-related and Fc receptor-related signaling. We aimed to investigate the impact of therapeutic intervention with HM71224 (LY3337641), a selective BTK inhibitor, on the development of murine SLE-like disease features. METHODS: We examined the therapeutic effects of HM71224 on SLE-like disease features in MRL/lpr and NZB/W F1 mice. The disease-related skin lesion was macroscopically observed in MRL/lpr mice, and the impact on splenomegaly and lymphadenopathy was determined by the weight of the spleen and cervical lymph node. The renal function was evaluated by measuring blood urea nitrogen, serum creatinine, and urine protein, and the renal damage was assessed by histopathological grading. Survival rate was observed during the administration period. The impact of B cell inhibition was investigated in splenocytes from both mice using flow cytometry. Autoantibody was measured in serum by ELISA. RESULTS: HM71224 effectively suppressed splenic B220+GL7+, B220+CD138+, and B220+CD69+ B cell counts, and anti-dsDNA IgG and reduced splenomegaly and lymph node enlargement. The compound also prevented skin lesions caused by lupus development, ameliorated renal inflammation and damage with increased blood urea nitrogen and creatinine, and decreased proteinuria. Furthermore, HM71224 also decreased mortality from lupus development in both mouse models. CONCLUSION: Our results indicate that inhibition of BTK by HM71224 effectively reduced B cell hyperactivity and significantly attenuated the development of SLE and LN in rodent SLE models.

HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.

BACKGROUND: Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis. METHODS: The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA). RESULTS: HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA. CONCLUSIONS: HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

Synthesis and biological evaluation of pyrimidine-based dual inhibitors of human epidermal growth factor receptor 1 (HER-1) and HER-2 tyrosine kinases.

A novel series of N(4)-(3-chlorophenyl)-5-(oxazol-2-yl)pyrimidine-4,6-diamines were synthesized and evaluated as dual inhibitors of HER-1/HER-2 tyrosine kinases. In contrast to the currently approved HER-2-targeted agent (lapatinib, 1), our irreversible HER-1/HER-2 inhibitors have the potential to overcome the clinically relevant and mutation-induced drug resistance. The selected compound (19a) showed excellent inhibitory activity toward HER-1/HER-2 tyrosine kinases with selectivity over 20 other kinases and inhibited the proliferation of both cancer cell types: lapatinib-sensitive cell lines (SK-Br3, MDA-MB-175, and N87) and lapatinib-resistant cell lines (MDA-MB-453, H1781, and H1975). The excellent pharmacokinetic profiles of 19a in mice and rats led us to further investigation of a novel therapeutic agent for HER-2-targeting treatment of solid tumors, especially HER-2-positive breast/gastric cancer and HER-2-mutated lung cancer.

Oncogenic Ras signals through activation of both phosphoinositide 3-kinase and Rac1 to induce c-Jun NH2-terminal kinase-mediated, caspase-independent cell death.

Cells avert the development of malignancy in response to deregulated oncogene expression by activating a regulated cell death pathway. However, the molecular mechanism underlying this oncogene-induced cellular death process remains unclear. Here, we show that retroviral expression of oncogenic H-ras induced cell death in a caspase-independent manner in normal cells. Inhibition of c-Jun NH2-terminal kinase (JNK) by pretreatment with SP600125 or a dominant-negative form of JNK blocked cell death. Rac1 and phosphoinositide 3-kinase (PI3K) were activated in cells overexpressing oncogenic H-ras. Inhibition of Rac1 with RacN17, a dominant-negative form of Rac1, attenuated oncogenic H-ras-induced JNK activation and subsequent cell death. Interestingly, inhibition of PI3K with LY294002 or by small interfering RNA-mediated knockdown of PI3K p85 or p110 subunits also clearly attenuated JNK activation and cell death. No cross talk was observed between Rac1 and PI3K, indicating that these pathways operate in parallel. Our findings show that JNK is necessary for oncogenic H-ras-induced, caspase-independent cell death, and that both PI3K and Rac1 activities are required for JNK activation and cell death. Determining the molecular mechanisms that mediate cell death responses to deregulated oncogenes provides a more refined understanding of cellular disposal processes in normal cells and increases our appreciation of these events as a mechanism for protecting against malignant progression.