McCormick, Daniel

Abstract Macropinocytosis supports the metabolic requirement of RAS‐transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS‐transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin‐binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2‐deletion construct, we demonstrate that the UBA‐domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP‐bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS‐driven cancers.

Abstract Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P<0.05 vs. BL. Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.

AbstractWe determined that the molecular defect of 2 patients with hemoglobin (Hb) M-Kankakee [Hb M-Iwate, 87 (F8) His → Tyr] resides in the 1-globin gene. The proportion of Hb M observed is higher than that predicted for an 1-globin variant. Our evidence suggests that the greater-than-expected proportion of Hb M-Kankakee results from preferential association of the electronegative β-globin chains with the M-globin chains that are more electropositive than normal -globin chains.

AbstractPrevious studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

We determined that the molecular defect of 2 patients with hemoglobin (Hb) M-Kankakee [Hb M-Iwate, 87 (F8) His → Tyr] resides in the 1-globin gene. The proportion of Hb M observed is higher than that predicted for an 1-globin variant. Our evidence suggests that the greater-than-expected proportion of Hb M-Kankakee results from preferential association of the electronegative β-globin chains with the M-globin chains that are more electropositive than normal -globin chains.

Despite optimal current therapies, cardiovascular disease remains the leading cause for death worldwide. Importantly, advances in peptide engineering have accelerated the development of innovative therapeutics for diverse human disease states. Additionally, the advancement of bispecific therapeutics targeting >1 signaling pathway represents a highly innovative strategy for the treatment of cardiovascular disease. We, therefore, engineered a novel, designer peptide, which simultaneously targets the pGC-A (particulate guanylyl cyclase A) receptor and the MasR (Mas receptor), potentially representing an attractive cardiorenoprotective therapeutic for cardiovascular disease. We engineered a novel, bispecific receptor activator, NPA7, that represents the fusion of a 22-amino acid sequence of BNP (B-type natriuretic peptide; an endogenous ligand of pGC-A) with Ang 1–7 (angiotensin 1–7)—the 7-amino acid endogenous activator of MasR. We assessed NPA7’s dual receptor activating actions in vitro (second messenger production and receptor interaction). Further, we performed an intravenous peptide infusion comparison study in normal canines to study its biological actions in vivo, including in the presence of an MasR antagonist. Our in vivo and in vitro studies demonstrate the successful synthesis of NPA7 as a bispecific receptor activator targeting pGC-A and MasR. In normal canines, NPA7 possesses enhanced natriuretic, diuretic, systemic, and renal vasorelaxing and cardiac unloading properties. Importantly, NPA7’s actions are superior to that of the individual native pGC-A or MasR ligands. These studies advance NPA7 as a novel, bispecific designer peptide with potential cardiorenal therapeutic benefit for the treatment of cardiovascular disease, such as hypertension and heart failure.

Abstract Susceptibility and resistance to experimental autoimmune thyroiditis is encoded by MHC H2A genes. We reported that traditionally resistant B10 (H2b) mice permit thyroiditis induction with mouse thyroglobulin (mTg) after depleting regulatory T cells (Tregs), supporting Ab presentation to thyroiditogenic T cells. Yet, Eak transgenic mice, expressing Ab and normally absent Eb molecules (E+B10 mice), are susceptible to thyroiditis induction without Treg depletion. To explore the effect of Eb expression on mTg presentation by Ab, seven putative Ab-binding, 15–16-mer peptides were synthesized. Five were immunogenic for both B10 and E+B10 mice. The effect of Eb expression was tested by competition with an Eα52-68 peptide, because Eα52-68 occupies ∼15% of Ab molecules in E+B10 mice, binding with high affinity. Eα52-68 competitively reduced the proliferative response to mTg, mTg1677, and mTg2342 of lymph node cells primed to each Ag. Moreover, mTg1677 induced mild thyroiditis in Treg-depleted B10 mice, and in E+B10 mice without the need for Treg depletion. Eα52-68 competition with mTg-derived peptides may impede clonal deletion of pathogenic, mTg-specific T cells in the thymus.