Kathleen L. King

Disruption of the Jak1 Gene Demonstrates Obligatory and Nonredundant Roles of the Jaks in Cytokine-Induced Biologic Responses

Herein we report the generation of mice lacking the ubiquitously expressed Janus kinase, Jak1. Jak1-/- mice are runted at birth, fail to nurse, and die perinatally. Although Jak1-/- cells are responsive to many cytokines, they fail to manifest biologic responses to cytokines that bind to three distinct families of cytokine receptors. These include all class II cytokine receptors, cytokine receptors that utilize the gamma(c) subunit for signaling, and the family of cytokine receptors that depend on the gp130 subunit for signaling. Our results thus demonstrate that Jak1 plays an essential and nonredundant role in promoting biologic responses induced by a select subset of cytokine receptors, including those in which Jak utilization was thought to be nonspecific.

The glycotope-specific RAV12 monoclonal antibody induces oncosis in vitro and has antitumor activity against gastrointestinal adenocarcinoma tumor xenografts in vivo

RAV12 is a chimeric antibody that recognizes an N-linked carbohydrate antigen (RAAG12) strongly expressed on multiple solid organ cancers. More than 90% of tumors of colorectal, gastric, and pancreatic origin express RAAG12, and a majority of these tumors exhibit uniform RAAG12 expression. RAV12 exhibits potent cytotoxic activity in vitro against COLO 205 colon tumor cells via an oncotic cell death mechanism. RAV12-treated COLO 205 cells undergo morphologic changes consistent with oncosis, including cytoskeletal rearrangement, rapid plasma membrane swelling, and cell lysis. RAV12 inhibited the growth of RAAG12-expressing gastrointestinal tumor xenografts in athymic mice. In the case of SNU-16 tumor cells, twice weekly treatment of established s.c. tumors with 10 mg/kg RAV12 caused a ∼70% suppression of tumor growth at the end of the study. This preclinical data has led to the initiation of a phase I/IIA clinical study of RAV12 in patients with metastatic or recurrent adenocarcinoma. [Mol Cancer Ther 2007;6(3):856–65]

Phenylephrine, endothelin, prostaglandin F2α, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro, and leukemia inhibitory factor induce different cardiac hypertrophy phenotypes in vitro

In these studies, we show that endothelin (ET), leukemia inhibitory factor (LIF), phenylephrine (PE), and prostaglandin F2α(PGF2α), which are all hypertrophic for neonatal rat cardiac myocytes in culture, induce distinct morphological, physiological, and genetic changes after a 48-h treatment. Transmission electron microscopy revealed differences in myofibril organization, with ET-treated cells containing the most mature-looking myofibrils and PGF2α — and LIF-treated cells the least. ET- and PE-treated cultures contained the same number of beating cells as control, but LIF and PGF2α treatment increased the number of beating cells 180%. Treatment with LIF, PE, and PGF2α increased the beat rate to 3.3 times that of control. After exposure to the β-adrenergic agonist isoproterenol, the beat rate increased 50% for PGF2α, 54% for PE, 84% for LIF, and 125% for control. ET treatment did not increase the beat rate, nor did these cells respond to isoproterenol. ET, LIF, and PE increased the production of atrial natriuretic peptide (ANP) by three-fold and PGF2α by 18-fold over nontreated cells. Brain natriuretic peptide (BNP) was increased fourfold by ET and PE, 16-fold by LIF, and 29-fold by PGF2α. Interestingly, on a pmol/L basis, only LIF induced more BNP than ANP. Treatment with all agents led to a similar pattern of gene induction: increased expression of the embryonic genes for ANP and skeletal α-actin, and less than a twofold change in the constitutively expressed gene myosin light chain-2, with the exception that LIF did not induce skeletal α-actin. Each agent, however, induced ANP mRNA with a different time-course. We conclude that at least four distinct cardiac myocyte hypertrophy response programs can be induced in vitro. Further studies are necessary to determine whether these correlate to the different types of cardiac hypertrophy seen in vivo.

Cardiac fibroblasts produce leukemia inhibitory factor and endothelin, which combine to induce cardiac myocyte hypertrophy in vitro.

Cardiac fibroblasts in culture produce factor(s) that induce hypertrophy of neonatal rat ventricular myocytes in vitro. As in vivo, the myocyte hypertrophy response in culture is characterized by an increase in cell size and contractile protein content, and by the activation of embryonic genes, including the gene for atrial natriuretic peptide. The purpose of this study was to identify the factor(s) produced by fibroblasts that induce myocyte hypertrophy. The fibroblast hypertrophy activity was inhibited using a combination of the endothelin A receptor blocker BQ-123 and an antibody to leukemia inhibitory factor. The individual antagonists each caused a partial inhibition. The mRNAs for both leukemia inhibitory factor and endothelin were detected by RT-PCR analysis and the concentration of both proteins was determined to be approximately 200 pmol/L in the conditioned medium using immunoassays. Purified leukemia inhibitory factor and endothelin each induced distinctive morphological changes in the myocytes. Their combination generated a different morphology similar to that induced by fibroblast conditioned medium. Each factor also induced atrial natriuretic peptide production, but both were required for the myocytes to produce the levels measured after exposure to fibroblast conditioned medium. These results show that hypertrophy activity produced by cardiac fibroblasts in culture is a result of leukemia inhibitory factor and endothelin.

Development of MGD007, a gpA33 x CD3 bispecific DART® protein for T-cell immunotherapy of metastatic colorectal cancer

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 μg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 μg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761–72. ©2018 AACR.

Endogenous cardiac vasoactive factors modulate endothelin production by cardiac fibroblasts in culture.

Endothelin, a potent vasoconstrictor, is produced by cardiac fibroblasts in culture and induces hypertrophy in cardiac myoctes. The purpose of this study was to determine whether vasoactive factors endogenous to the heart affect the production of endothelin by cultured cardiac fibroblasts. Vasoactive factors have been shown to play multiple roles in the adaptation of the heart to chronic overload, affecting both vascular tone and cell growth. Both atrial (ANP) and brain (BNP) natriuretic peptides are endogenous cardiac vasodilators and are produced by cultured myocytes in response to stimulation with endothelin. Treatment of cardiac fibroblasts with these peptides decreased endothelin production. Nitroprusside, an activator of guanylyl cyclase, decreased endothelin production indicating the involvement of cGMP in the response. Carbaprostacyclin, a stable derivative of prostacyclin, another endogenous cardiac vasodilator, also decreased endothelin production by fibroblasts. The combination of BNP and carbaprostacyclin was additive in decreasing endothelin production. In contrast, PGF2α and angiotensin II, both endogenous cardiac vasoconstrictors, increased endothelin production and overcame the inhibition induced by BNP and carba-prostacyclin. In summary, endothelin production by cardiac fibroblasts was decreased by the endogenous cardiac vasodilators ANP, BNP, and prostacyclin and increased by the endogenous vasoconstrictors PGF2α and angiotensin II.