Timing of anti-PD-L1 antibody initiation affects efficacy/toxicity of CD19 CAR T-cell therapy for large B-cell lymphoma.

ABSTRACT: More than half of the patients treated with CD19-targeted chimeric antigen receptor (CAR) T-cell immunotherapy for large B-cell lymphoma (LBCL) do not achieve durable remission, which may be partly due to PD-1/PD-L1-associated CAR T-cell dysfunction. We report data from a phase 1 clinical trial (NCT02706405), in which adults with LBCL were treated with autologous CD19 CAR T cells (JCAR014) combined with escalating doses of the anti-PD-L1 monoclonal antibody, durvalumab, starting either before or after CAR T-cell infusion. The addition of durvalumab to JCAR014 was safe and not associated with increased autoimmune or immune effector cell-associated toxicities. Patients who started durvalumab before JCAR014 infusion had later onset and shorter duration of cytokine release syndrome and inferior efficacy, which was associated with slower accumulation of CAR T cells and lower concentrations of inflammatory cytokines in the blood. Initiation of durvalumab before JCAR014 infusion resulted in an early increase in soluble PD-L1 (sPD-L1) levels that coincided with the timing of maximal CAR T-cell accumulation in the blood. In vitro, sPD-L1 induced dose-dependent suppression of CAR T-cell effector function, which could contribute to inferior efficacy observed in patients who received durvalumab before JCAR014. Despite the lack of efficacy improvement and similar CAR T-cell kinetics early after infusion, ongoing durvalumab therapy after JCAR014 was associated with re-expansion of CAR T cells in the blood, late regression of CD19+ and CD19- tumors, and enhanced duration of response. Our results indicate that the timing of initiation of PD-L1 blockade is a key variable that affects outcomes after CD19 CAR T-cell immunotherapy for adults with LBCL.

Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Role of Smad3 in platelet-derived growth factor-C-induced liver fibrosis.

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-ß (TGFß) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFß signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFß/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFß signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor-stromal interactions in the liver.

A random mutation capture assay to detect genomic point mutations in mouse tissue.

Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.

Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice.

UNLABELLED: Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. CONCLUSION: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC.

The Nck-interacting kinase phosphorylates ERM proteins for formation of lamellipodium by growth factors.

The mammalian Ste20-like Nck-interacting kinase (NIK) and its orthologs Misshapen in Drosophila and Mig-15 in Caenorhabditis elegans have a conserved function in regulating cell morphology, although through poorly understood mechanisms. We report two previously unrecognized actions of NIK: regulation of lamellipodium formation by growth factors and phosphorylation of the ERM proteins ezrin, radixin, and moesin. ERM proteins regulate cell morphology and plasma membrane dynamics by reversibly anchoring actin filaments to integral plasma membrane proteins. In vitro assays show that NIK interacts directly with ERM proteins, binding their N termini and phosphorylating a conserved C-terminal threonine. In cells, NIK and phosphorylated ERM proteins localize at the distal margins of lamellipodia, and NIK activity is necessary for phosphorylation of ERM proteins induced by EGF and PDGF, but not by thrombin. Lamellipodium extension in response to growth factors is inhibited in cells expressing a kinase-inactive NIK, suppressed for NIK expression with siRNA oligonucleotides, or expressing ezrin T567A that cannot be phosphorylated. These data suggest that direct phosphorylation of ERM proteins by NIK constitutes a signaling mechanism controlling growth factor-induced membrane protrusion and cell morphology.

The STE20 kinase HGK is broadly expressed in human tumor cells and can modulate cellular transformation, invasion, and adhesion.

HGK (hepatocyte progenitor kinase-like/germinal center kinase-like kinase) is a member of the human STE20/mitogen-activated protein kinase kinase kinase kinase family of serine/threonine kinases and is the ortholog of mouse NIK (Nck-interacting kinase). We have cloned a novel splice variant of HGK from a human tumor line and have further identified a complex family of HGK splice variants. We showed HGK to be highly expressed in most tumor cell lines relative to normal tissue. An active role for this kinase in transformation was suggested by an inhibition of H-Ras(V12)-induced focus formation by expression of inactive, dominant-negative mutants of HGK in both fibroblast and epithelial cell lines. Expression of an inactive mutant of HGK also inhibited the anchorage-independent growth of cells yet had no effect on proliferation in monolayer culture. Expression of HGK mutants modulated integrin receptor expression and had a striking effect on hepatocyte growth factor-stimulated epithelial cell invasion. Together, these results suggest an important role for HGK in cell transformation and invasiveness.