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CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.

Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment., Significance: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950., (©2024 American Association for Cancer Research.)

Treatment for myelodysplastic syndromes (MDS) remains insufficient due to clonal heterogeneity and lack of effective clinical therapies. Dysregulation of apoptosis is observed across MDS subtypes regardless of mutations and represents an attractive therapeutic opportunity. Venetoclax (VEN), a selective inhibitor of anti-apoptotic protein B-cell lymphoma- 2 (BCL2), has yielded impressive responses in older patients with acute myeloid leukemia (AML) and high risk MDS. BCL2 family anti-apoptotic proteins BCL-XL and induced myeloid cell leukemia 1 (MCL1) are implicated in leukemia survival, and upregulation of MCL1 is seen in VEN-resistant AML and MDS. We determined in vitro sensitivity of MDS patient samples to selective inhibitors of BCL2, BCL-XL and MCL1. While VEN response positively correlated with MDS with excess blasts, all MDS subtypes responded to MCL1 inhibition. Treatment with combined VEN + MCL1 inhibtion was synergistic in all MDS subtypes without significant injury to normal hematopoiesis and reduced MDS engraftment in MISTRG6 mice, supporting the pursuit of clinical trials with combined BCL2 + MCL1 inhibition in MDS.

Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome characterized by monocytic proliferation in the presence of dysplastic bone marrow changes, inflammatory symptoms, and propensity for transformation to acute myeloid leukemia (AML), with a poor prognosis and limited treatment options. Unlike the α and β isoforms, the phosphatidylinositol-3-kinase (PI3K)-δ signaling protein is predominantly expressed by hematopoietic cells and therefore has garnered interest as a potential target for the treatment of lymphomas and leukemias. We revealed a pattern of increased PIK3CD:PIK3CA ratio in monocytic M5 AML patients and cell lines, and this ratio correlated with responsiveness to pharmacological PI3K-δ inhibition in vitro. Because CMML is a disease defined by monocytic clonal proliferation, we tested the PI3K-δ inhibitor umbralisib as a single agent and in combination with the JAK1/2 inhibitor ruxolitinib, in CMML. Our ex vivo experiments with primary CMML patient samples revealed synergistic inhibition of viability and clonogenicity with this combination. Phospho-specific flow cytometry revealed that dual inhibition had the unique ability to decrease STAT5, ERK, AKT, and S6 phosphorylation simultaneously, which offers a mechanistic hypothesis for the enhanced efficacy of the combination treatment. These preclinical data indicate promising activity by co-inhibition of PI3K-δ and JAK1/2 and support the use of ruxolitinib + umbralisib combination therapy in CMML under active clinical investigation., Competing Interests: Conflict of interest disclosure MRS receives research funding from Astex, Incyte, Millennium, and TG Therapeutics; serves on consultancy/advisory board/monitoring committees for AbbVie, Astex, BMS, Celgene, Geron, Karyopharm, Millennium, Ryvu, Sunesis, and TG Therapeutics; has equity in Karyopharm; and has patents and royalties with Boehringer-Ingelheim. The remaining authors have no competing financial interests., (Copyright © 2021. Published by Elsevier Inc.)

Purpose: The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients. Bromodomain and extraterminal inhibitors (BETi) are potential treatments for AML, as regulators of critical AML oncogenes. We tested the efficacy of novel BET inhibitor INCB054329, and its synergy with venetoclax to reduce AML without induction of hematopoietic toxicity., Experimental Design: INCB054329 efficacy was assessed by changes in cell cycle and apoptosis in treated AML cell lines. In vivo efficacy was assessed by tumor reduction in MV-4-11 cell line-derived xenografts. Precision run-on and sequencing (PRO-seq) evaluated effects of INCB054329. Synergy between low-dose BETi and venetoclax was assessed in cell lines and patient samples in vitro and in vivo while efficacy and toxicity was assessed in patient-derived xenograft (PDX) models., Results: INCB054329 induced dose-dependent apoptosis and quiescence in AML cell lines. PRO-seq analysis evaluated the effects of INCB054329 on transcription and confirmed reduced transcriptional elongation of key oncogenes, MYC and BCL2 , and genes involved in the cell cycle and metabolism. Combinations of BETi and venetoclax led to reduced cell viability in cell lines and patient samples. Low-dose combinations of INCB054329 and venetoclax in cell line and PDX models reduced AML burden, regardless of the sensitivity to monotherapy without development of toxicity., Conclusions: Our findings suggest low dose combinations of venetoclax and BETi may be more efficacious for patients with AML than either monotherapy, potentially providing a longer, more tolerable dosing regimen., (©2020 American Association for Cancer Research.)

The selective inhibitor of nuclear export (SINE) compounds selinexor (KPT-330) and eltanexor (KPT-8602) are from a novel class of small molecules that target exportin-1 (XPO1 [CRM1]), an essential nucleo-cytoplasmic transport protein responsible for the nuclear export of major tumor suppressor proteins and growth regulators such as p53, p21, and p27. XPO1 also affects the translation of messenger RNAs for critical oncogenes, including MYC, BCL2, MCL1, and BCL6, by blocking the export of the translation initiation factor eIF4E. Early trials with venetoclax (ABT-199), a potent, selective inhibitor of BCL2, have revealed responses across a variety of hematologic malignancies. However, many tumors are not responsive to venetoclax. We used models of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) to determine in vitro and in vivo responses to treatment with venetoclax and SINE compounds combined. Cotreatment with venetoclax and SINE compounds demonstrated loss of viability in multiple cell lines. Further in vitro analyses showed that this enhanced cell death was the result of an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays, coinciding with activation of p53 and loss of MCL1. Treatment with SINE compounds and venetoclax combined led to a reduction in tumor growth in both AML and DLBCL xenografts. Immunohistochemical analysis of tissue sections revealed that the reduction in tumor cells was partly the result of an induction of apoptosis. The enhanced effects of this combination were validated in primary AML and DLBCL patient cells. Our studies reveal synergy with SINE compounds and venetoclax in aggressive hematologic malignancies and provide a rationale for pursuing this approach in a clinical trial., (© 2020 by The American Society of Hematology.)

Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials. SIGNIFICANCE: Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML. See related commentary by Leber et al., p. 1511 . This article is highlighted in the In This Issue feature, p. 1494 ., (©2018 American Association for Cancer Research.)

DNA replication stress promotes genome instability in cancer. However, the contribution of the replication stress response to the development of malignancies remains unresolved. The DNA replication stress response protein SMARCAL1 stabilizes DNA replication forks and prevents replication fork collapse, a cause of DNA breaks and apoptosis. While the fork regression/remodeling functions of SMARCAL1 have been investigated, its in vivo functions in replication stress and cancer are unclear. Using a gamma radiation (IR)-induced replication stress T-cell lymphoma mouse model, we observed a significant inhibition of lymphomagenesis in mice lacking one or both alleles of Smarcal1. Notably, a quarter of the Smarcal1-deficient mice did not develop tumors. Moreover, hematopoietic stem/progenitor cells (HSPCs) and developing thymocytes in Smarcal1-deficient mice showed increased DNA damage and apoptosis during the proliferation burst following IR and an impaired ability to repopulate the thymus after IR. Additionally, mice lacking Smarcal1 showed significant HSPC defects when challenged to respond to other replication stress stimuli. Thus, our data reveal the critical function of the DNA replication stress response and, specifically, Smarcal1 in hematopoietic cell survival and tumor development. Our results also provide important insight into the immunodeficiency observed in individuals with mutations in SMARCAL1 by suggesting that it is an HSPC defect.

MicroRNA (miR) are important regulators of gene expression, and aberrant miR expression has been linked to oncogenesis; however, little is understood about their contribution to lung tumorigenesis. Here, we determined that miR-31 is overexpressed in human lung adenocarcinoma and this overexpression independently correlates with decreased patient survival. We developed a transgenic mouse model that allows for lung-specific expression of miR-31 to test the oncogenic potential of miR-31 in the lung. Using this model, we observed that miR-31 induction results in lung hyperplasia, followed by adenoma formation and later adenocarcinoma development. Moreover, induced expression of miR-31 in mice cooperated with mutant KRAS to accelerate lung tumorigenesis. We determined that miR-31 regulates lung epithelial cell growth and identified 6 negative regulators of RAS/MAPK signaling as direct targets of miR-31. Our study distinguishes miR-31 as a driver of lung tumorigenesis that promotes mutant KRAS-mediated oncogenesis and reveals that miR-31 directly targets and reduces expression of negative regulators of RAS/MAPK signaling.

The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the 13 different cancer types evaluated, 6-90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx-inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage-response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

Despite its involvement in most human cancers, MYC continues to pose a challenge as a readily tractable therapeutic target. Here we identify the MYC transcriptional cofactors TIP48 and TIP49 and MYC as novel binding partners of Mdm2-binding protein (MTBP), a functionally undefined protein that we show is oncogenic and overexpressed in many human cancers. MTBP associated with MYC at promoters and increased MYC-mediated transcription, proliferation, neoplastic transformation, and tumor development. In breast cancer specimens, we determined overexpression of both MYC and MTBP was associated with a reduction in 10-year patient survival compared with MYC overexpression alone. MTBP was also frequently co-amplified with MYC in many human cancers. Mechanistic investigations implicated associations with TIP48/TIP49 as well as MYC in MTBP function in cellular transformation and the growth of human breast cancer cells. Taken together, our findings show MTBP functions with MYC to promote malignancy, identifying this protein as a novel general therapeutic target in human cancer., (©2014 American Association for Cancer Research.)

Many tumor cells express globally reduced levels of microRNAs (miRNA), suggesting that decreased miRNA expression in premalignant cells contributes to their tumorigenic phenotype. In support of this, Dicer, an RNase III-like enzyme that controls the maturation of miRNA, was recently shown to function as a haploinsufficient tumor suppressor in nonhematopoietic cells. Because the Myc oncoprotein, a critical inducer of B-cell lymphomas, was reported to suppress the expression of multiple miRNAs in lymphoma cells, it was presumed that a deficiency of Dicer and subsequent loss of miRNA maturation would accelerate Myc-induced lymphoma development. We report here that, surprisingly, a haploinsufficiency of Dicer in B cells failed to promote B-cell malignancy or accelerate Myc-induced B-cell lymphomagenesis in mice. Moreover, deletion of Dicer in B cells of CD19-cre(+)/Emicro-myc mice significantly inhibited lymphomagenesis, and all lymphomas that did arise in these mice lacked functional Cre expression and retained at least one functional Dicer allele. Uncharacteristically, the lymphomas that frequently developed in the CD19-cre(+)/Dicer(fl/fl)/Emicro-myc mice were of very early precursor B-cell origin, a stage of B-cell development prior to Cre expression. Therefore, loss of Dicer function was not advantageous for lymphomagenesis, but rather, Dicer ablation was strongly selected against during Myc-induced B-cell lymphoma development. Moreover, deletion of Dicer in established B-cell lymphomas resulted in apoptosis, revealing that Dicer is required for B-cell lymphoma survival. Thus, Dicer does not function as a haploinsufficient tumor suppressor in B cells and is required for B-cell lymphoma development and survival., ((c)2010 AACR.)

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Emu-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis.

Transcription factor NF-kappaB controls the expression of multiple genes involved in immunity and inflammation. The initial activation and duration of NF-kappaB signaling is regulated by posttranslational modifications to IkappaB kinase, which earmarks inhibitors of NF-kappaB for degradation. Prior studies suggest that K63-linked ubiquitination of NEMO (NF-kappaB essential modulator), an IkappaB kinase regulatory subunit, is critical for NF-kappaB and MAPK signaling following engagement of Ag receptors. We now demonstrate that NF-kappaB and MAPK pathways are largely unaffected in primary cells from mice harboring a ubiquitination-defective form of NEMO, NEMO-KR. TLR- but not Ag receptor-induced cellular responses are impaired in NEMO-KR mice, which are more resistant to LPS-induced endotoxic shock than wild-type animals. Thus, one function of NEMO ubiquitination is to fine tune innate immune responses under TLR control.

We have previously reported that junctional adhesion molecule 2 (JAM2) adheres to T cells through heterotypic interactions with JAM3. An examination of the cation dependence of JAM2 adhesion to HSB cells revealed a Mn(2+)-enhanced binding component indicative of integrin involvement. Using neutralizing integrin antibodies, we have defined an interaction between JAM2 and alpha(4)beta(1) in T cells. The interaction is readily amenable to drug intervention as demonstrated by the ability of TBC 772, an alpha(4)-specific inhibitor, to attenuate the Mn(2+)-enhanced component. Intriguingly, the engagement of alpha(4)beta(1) by JAM2 is only enabled following prior adhesion of JAM2 with JAM3 and is not detectable in cells where JAM3 expression is absent. Supporting this observation, we show that neutralizing JAM3 serum and soluble JAM3 ectodomain inhibit not only JAM2 binding to JAM3 but also prevent JAM2/alpha(4)beta(1) interactions in T cells. We further define the first Ig-like fold of JAM2 as being competent in binding both JAM3 and alpha(4)beta(1) counter-receptors. Mutagenesis of the only acidic residue in the C-D loop of this Ig fold, namely Asp-82, has no bearing on alpha(4)beta(1) interactions, and thus JAM2 deviates somewhat from the mechanism used by other immunoglobulin superfamily cell adhesion molecules to engage integrin.

We have identified a third member of the junctional adhesion molecule (JAM) family. At the protein level JAM3 displays 36 and 32% identity to JAM2 and JAM1, respectively. The coding region is distributed over 9 exons and maps to chromosome 11q25. The gene shows widespread tissue expression with higher levels apparent in the kidney, brain, and placenta. At the cellular level we show expression of JAM3 transcript within endothelial cells. Our major finding is that JAM3 and JAM2 are binding partners. Thus, JAM3 ectodomain binds firmly to JAM2-Fc. This heterotypic interaction is maintained when JAM3-Fc is used to capture Chinese hamster ovary cells expressing full-length JAM2. In static adhesion assays we show that JAM3 is unable to bind to leukocyte cell lines. This is consistent with the lack of JAM2 expression. However, using JAM2-Fc pull-down experiments in combination with polyclonal anti-JAM3 serum, we demonstrate that JAM3 is the previously uncharacterized 43-kDa counter-receptor that mediates JAM2 adhesion to T cells. Most significantly we demonstrate up-regulation of JAM3 protein on peripheral blood lymphocytes following activation. Finally we show the utility of JAM3 ectodomain as an inhibitor of JAM2 adhesion.

We have cloned a novel cDNA belonging to the Ig superfamily that shows 44% similarity to the junctional adhesion molecule (JAM) and maps to chromosome 21q21.2. The open reading frame of JAM2 predicts a 34-kDa type I integral membrane protein that features two Ig-like folds and three N-linked glycosylation sites in the extracellular domain. A single protein kinase C phosphorylation consensus site and a PDZ-binding motif are present in the short intracellular tail. Heterologous expression of JAM2 in Chinese hamster ovary cells defined a 48-kDa protein that localizes predominantly to the intercellular borders. Northern blot analysis showed that JAM2 is preferentially expressed in the heart. JAM2 homotypic interactions were demonstrated by the ability of JAM2-Fc to capture JAM2-expressing Chinese hamster ovary cells. We further showed that JAM2, but not JAM1, is capable of adhering to the HSB and HPB-ALL lymphocyte cell lines. Neutralizing mouse anti-JAM2 polyclonal antibodies provided evidence against homotypic interactions in this assay. Biotinylation of HSB cell membranes revealed a 43-kDa counter-receptor that precipitates specifically with JAM2-Fc. These characteristics of JAM2 led us to hypothesize a role for this novel protein in adhesion events associated with cardiac inflammatory conditions.

The kinase insert domain-containing receptor (KDR) tyrosine kinase mediates calcium mobilization in endothelial cells and plays a key role during physiological and pathological angiogenesis. To provide a detailed understanding of how KDR is activated, we analyzed the kinetics of ligand-receptor interaction using BIAcore. Both predimerized (KDR-Fc) and monomeric (KDR-cbu) receptors were examined with vascular endothelial cell growth factor (VEGF) homodimers and VEGF/placental growth factor (PlGF) heterodimers. VEGF binds to KDR-Fc with ka = 3.6 +/- 0.07e6, kd = 1.34 +/- 0.19e-4, and KD = 37.1 +/- 4.9 pM. These values are similar to those displayed by monomeric KDR where ka = 5.23 +/- 1.4e6, kd = 2.74 +/- 0.76e-4, and KD = 51.7 +/- 5.8 pM were apparent. In contrast, VEGF/PlGF bound to KDR-Fc with ka = 7.3 +/- 1.6e4, kd = 4.4 +/- 1. 2e-4, and KD = 6 +/- 1.2 nM. Thus, the heterodimer displays a 160-fold reduced KD for binding to predimerized KDR, which is mainly a consequence of a 50-fold reduction in ka. We were unable to detect association between VEGF/PlGF and monomeric KDR. However, nanomolar concentrations of VEGF/PlGF were able to elicit weak calcium mobilization in endothelial cells. This latter observation may indicate partial predimerization of KDR on the cell surface or facilitation of binding due to accessory receptors.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.

Vascular endothelial cell growth factor interacts with the receptor tyrosine kinases Flt-1 and KDR/Flk-1. We report that both receptors bind to PLC gamma and display specificity for the N-SH2 over the C-SH2 domain. Extensive site-directed mutagenesis of Flt-1 reveals that the juxta-membrane Y794, and the carboxyl terminal Y1169, are two major sites of interaction. Amino acids in the +1, +2 and +3 positions following these tyrosines are LSI and IPI, respectively. Peptide maps generated from wild type and mutant Flt-1 confirms that these residues are autophosphorylated. As predicted, mutagenesis of the analogous amino acids in KDR, positions Y801F and Y1175F, which lie in contexts YLSI and YIVL, respectively, reduced interactions of PLC gamma with this receptor. We conclude that both Flt-1 and KDR have the potential to signal through PLC gamma via phosphotyrosine residues located in juxta-membrane and carboxyl tail regions.

Vascular Endothelial Growth Factor (VEGF) mediates its actions through the Flt-1 and KDR(Flk-1) receptor tyrosine kinases. To localize the extracellular region of Flt-1 that is involved in ligand interactions, we prepared secreted fusion proteins between various combinations of its seven extracellular IgG-like folds. Ligand binding studies show that in combination, domains one and two (amino acids 1-234) are sufficient to achieve VEGF165 interactions. Either domain alone is insufficient to achieve this effect. However, Scatchard analysis reveals that despite the binding capabilities of this construct, the Kd is five fold lower than ligand binding to the full extracellular domain. We find that addition of domain three to this minimal site restores high affinity receptor binding. Further, we show that domains one and two are sufficient to achieve interactions of Flt-1 with Placental Growth Factor (PIGF-1).

We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.

A bovine homologue of the rat and human epithelial Na+ channel subunits, alpha-rENaC and alpha-hENaC, was cloned. The cDNA clone, termed alpha-bENaC, was isolated from a bovine renal papillary collecting duct cDNA expression library. The bovine cDNA is 3,584 base pairs (bp) long, has an open reading frame of 2,094 bp encoding a 697-amino acid protein, and is 75-85% homologous to its rat and human counterparts. In vitro translation of the transcribed cRNA yields an 80-kDa polypeptide and one at 92 kDa in the presence of pancreatic microsomes. The clone exhibits consensus sequences for N-linked glycosylation and for phosphorylation by protein kinase C, but not for protein kinase A. After expression in Xenopus laevis oocytes, a small amiloride-sensitive Na+ conductance that exhibited inward rectification and a reversal potential greater than +30 mV, consistent with the predicted equilibrium potential for Na+, was identified. The expressed alpha-bENaC-associated Na+ current was not responsive to elevations in adenosine 3',5'-cyclic monophosphate but could be stimulated by phorbol 12-myristate 13-acetate, an activator of protein kinase C. alpha-bENaC also formed amiloride-sensitive chimeric channels when coexpressed with the rat beta- and gamma-ENaC subunits in Xenopus oocytes. alpha-bENaC therefore represents a novel isoform of a growing family of epithelial Na+ channels.

Zn(II) is an essential trace element. In spermatozoa, Zn(II) modulates metabolism and chromatin condensation. The mechanisms of uptake and distribution of this ion in sperm cells have not been explored. In rat spermatids, our results indicate that 1) 65Zn(II) binds with fast kinetics to a labile, presumably extracellular, compartment. This binding is temperature insensitive and not modified by metabolic inhibitors. 2) Entry of 65Zn(II) in the absence of externally added proteins occurs through a mediated transport system that allows exchange to reach steady state in approximately 15 min at 34 degrees C. 3) Upon entering the cells, 65Zn(II) binds tightly to cellular organelles. 4) Exchangeable Zn(II) bound to cytoplasmic proteins plus free intracellular Zn(II) appears to be < 20% of total exchangeable Zn(II). 5) The intracellular exchangeable Zn(II) compartment is decreased by metabolic inhibitors, showing a direct or indirect link between energy metabolism and cellular Zn(II) levels. 6) 65Zn(II) efflux from rat spermatids is a process with a rate constant of 0.16 +/- 0.13 min-1 at 34 degrees C. This exit rate of Zn(II) is likely to be affected by Zn(II) release from cytoplasmic binding sites or organelles.

We characterized the electrophysiological properties of a chloride channel protein isolated from bovine trachea after incorporation into planar lipid bilayers, and studied the effects of thiol-modulating agents on channel regulation both in bilayers and vesicular iodide uptake studies. Our experiments showed that this protein formed perfectly anion-selective channels in the bilayer, with an anion permeability sequence of I- (2.1) > NO3- (1.7) > Br- (1.2) > Cl- (1.0). The conductance of this channel was 25-30 picosiemens in 150 mM Cl-, and saturated with increasing chloride concentration. This channel could be completely inhibited by 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate. Immunoblot analysis, using polyclonal antibodies (anti-p38), revealed one major band at 140 kDa. Upon reduction with dithiothreitol, 64- and 38-kDa polypeptides were observed. Functional experiments showed that reduction was accompanied by loss of 125I- uptake and single-channel activity. In the presence of dithiothreitol, only the low molecular mass protein forms (64 and 38 kDa) were detected by anti-p38 antibodies on Western blots. Cross-linking of S-S bonds with Cu(2+)-o-phenanthroline led to activation of chloride channels in vesicles and bilayers. Over-aggregation of chloride channels by this S-S cross-linking reagent caused inhibition of 125I- uptake by 80-100% and the abolishment of single-channel activity. We propose that the native chloride channel from bovine trachea can exist in vivo in different structural and functional forms depending upon its thiol-disulfide oxidation reduction status. The oxidized form has a molecular mass of 140 kDa and represents a fully active chloride channel. Inactivation of this channel might occur by over-aggregation of protein subunits, or by dissociation of the 140-kDa subunit by disulfide bond reduction.