α-PD-1 therapy elevates Treg/Th balance and increases tumor cell pSmad3 that are both targeted by α-TGFß antibody to promote durable rejection and immunity in squamous cell carcinomas.

BACKGROUND: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFß signaling. METHODS: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFß or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. RESULTS: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFß monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFß, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFß antibody administration attenuates these effects. We show that α-TGFß acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFß acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery. CONCLUSIONS: We show that α-PD-1 not only initiates a tumor rejection program, but can induce a competing TGFß-driven immuno-suppressive program. We identify new opportunities for α-PD-1/α-TGFß combinatorial treatment of SCCs especially those with a high mutation load, high CD4+ T cell content and pSmad3 signaling. Our data form the basis for clinical trial of α-TGFß/α-PD-1 combination therapy (NCT02947165).

MRI methods for evaluating the effects of tyrosine kinase inhibitor administration used to enhance chemotherapy efficiency in a breast tumor xenograft model.

PURPOSE: To evaluate whether quantitative MRI parameters are sensitive to the effects of the tyrosine kinase inhibitor gefitinib and can discriminate between two different treatment protocols. MATERIALS AND METHODS: Untreated mice with BT474 breast tumor xenografts were characterized in a preliminary study. Subsequently, tumor volume, apparent diffusion coefficient (ADC), transendothelial permeability (K(ps)), and fractional plasma volume (fPV) were measured in three groups of mice receiving: 1) control vehicle for 10 days, or gefitinib as 2) a single daily dose for 10 days or 3) a 2-day pulsed dose. RESULTS: Gefitinib treatment resulted in significant tumor growth inhibition (pulsed: 439 +/- 93; daily: 404 +/- 53; control: 891 +/- 174 mm(3), P < 0.050) and lower cell density (pulsed: 0.15 +/- 0.01, daily: 0.17 +/- 0.01, control: 0.24 +/- 0.01, P < 0.050) after 9 days. Tumor ADC increased in treated groups but decreased in controls (P > 0.050). Tumor K(ps) decreased with pulsed treatment but rebounded afterwards and increased with daily treatment (P > 0.050). Tumor fPV increased in both treated groups, decreasing afterwards with pulsed treatment (P > 0.050). CONCLUSION: Quantitative MRI can provide a sensitive measure of gefitinib-induced tumor changes, potentially distinguish between treatment regimens, and may be useful for determining optimal treatment scheduling for enhancing chemotherapy delivery.

Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015).

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM.

The human neurodegenerative and cancer predisposition condition ataxia-telangiectasia is characterized at the cellular level by radiosensitivity, chromosomal instability, and impaired induction of ionizing radiation-induced cell cycle checkpoint controls. Recent work has revealed that the gene defective in ataxia-telangiectasia, termed ATM, encodes an approximately 350-kDa polypeptide, ATM, that is a member of the phosphatidylinositol 3-kinase family. We show that ATM binds DNA and exploit this to purify ATM to near homogeneity. Atomic force microscopy reveals that ATM exists in two populations, with sizes consistent with monomeric and tetrameric states. Atomic force microscopy analyses also show that ATM binds preferentially to DNA ends. This property is similar to that displayed by the DNA-dependent protein kinase catalytic subunit, a phosphatidylinositol 3-kinase family member that functions in DNA damage detection in conjunction with the DNA end-binding protein Ku. Furthermore, purified ATM contains a kinase activity that phosphorylates serine-15 of p53 in a DNA-stimulated manner. These results provide a biochemical assay system for ATM, support genetic data indicating distinct roles for DNA-dependent protein kinase and ATM, and suggest how ATM may signal the presence of DNA damage to p53 and other downstream effectors.

The ataxia-telangiectasia related protein ATR mediates DNA-dependent phosphorylation of p53.

Levels of the tumour suppressor protein p53 are increased in response to a variety of DNA damaging agents. DNA damage-induced phosphorylation of p53 occurs at serine-15 in vivo. Phosphorylation of p53 at serine-15 leads to a stabilization of the polypeptide by inhibiting its interaction with Mdm2, a protein that targets p53 for ubiquitin-dependent degradation. However, the mechanisms by which DNA damage is signalled to p53 remain unclear. Here, we report the identification of a novel DNA-activated protein kinase that phosphorylates p53 on serine-15. Fractionation of HeLa nuclear extracts and biochemical analyses indicate that this kinase is distinct from the DNA-dependent protein kinase (DNA-PK) and corresponds to the human cell cycle checkpoint protein ATR. Immunoprecipitation studies of recombinant ATR reveal that catalytic activity of this polypeptide is required for DNA-stimulated phosphorylation of p53 on serine-15. These data suggest that ATR may function upstream of p53 in a signal transduction cascade initiated upon DNA damage and provide a biochemical assay system for ATR activity.

Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional expression.

The signal recognition particle (SRP) is an evolutionarily conserved ribonucleoprotein (RNP) complex that functions in protein targeting to the endoplasmic reticulum (ER) membrane. Only two protein subunits of the SRP, Srp54p and Sec65p, and the RNA subunit, scR1, were previously known in the yeast Saccharomyces cerevisiae. Purification of yeast SRP by immunoaffinity chromatography revealed five additional proteins. Amino acid sequencing and cloning of the genes encoding four of these proteins demonstrated that the yeast SRP contains homologs (termed Srp14p, Srp68p and Srp72p) of the SRP14, SRP68 and SRP72 subunits found in mammalian SRP. The yeast SRP also contains a 21 kDa protein (termed Srp21p) that is not homologous to any protein in mammalian SRP. An additional 7 kDa protein may correspond to the mammalian SRP9. Disruption of any one of the four genes encoding the newly identified SRP proteins results in slow cell growth and inefficient protein translocation across the ER membrane. These phenotypes are indistinguishable from those resulting from the disruption of genes encoding SRP components identified previously. These data indicate that a lack of any of the analyzed SRP components results in loss of SRP function. ScR1 RNA and SRP proteins are at reduced levels in cells lacking any one of the newly identified proteins. In contrast, SRP components are present at near wild type levels and SRP subparticles are present in cells lacking either Srp54p or Sec65p. Thus Srp14p, Srp21p, Srp68p and Srp72p, but not Sec65p or Srp54p, are required for stable expression of the yeast SRP.

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.

The signal recognition particle in S. cerevisiae.

We have identified the Saccharomyces cerevisiae homolog of the signal recognition particle (SRP) and characterized its function in vivo. S. cerevisiae SRP is a 16S particle that includes a homolog of the signal sequence-binding protein subunit of SRP (SRP54p) and a small cytoplasmic RNA (scR1). Surprisingly, the genes encoding scR1 and SRP54p are not essential for growth, though SRP-deficient cells grow poorly, suggesting that SRP function can be partially by-passed in vivo. Protein translocation across the ER membrane is impaired in SRP-deficient cells, indicating that yeast SRP, like its mammalian counterpart, functions in this process. Unexpectedly, the degree of the translocation defect varies for different proteins. The ability of some proteins to be efficiently targeted in SRP-deficient cells may explain why previous genetic and biochemical analyses in yeast and bacteria did not reveal components of the SRP-dependent protein targeting pathway.

Saccharomyces cerevisiae and Schizosaccharomyces pombe contain a homologue to the 54-kD subunit of the signal recognition particle that in S. cerevisiae is essential for growth.

We have isolated and sequenced genes from Saccharomyces cerevisiae (SRP54SC) and Schizosaccharomyces pombe (SRP54sp) encoding proteins homologous to both the 54-kD protein subunit (SRP54mam) of the mammalian signal recognition particle (SRP) and the product of a gene of unknown function in Escherichia coli, ffh (Römisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Nature (Lond.). 340:478-482; Bernstein H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, P. Walter. 1989. Nature (Lond.). 340:482-486). To accomplish this we took advantage of short stretches of conserved sequence between ffh and SRP54mam and used the polymerase chain reaction (PCR) to amplify fragments of the homologous yeast genes. The DNA sequences predict proteins for SRP54sc and SRP54sp that are 47% and 52% identical to SRP54mam, respectively. Like SRP54mam and ffh, both predicted yeast proteins contain a GTP binding consensus sequence in their NH2-terminal half (G-domain), and methionine-rich sequences in their COOH-terminal half (M-domain). In contrast to SRP54mam and ffh the yeast proteins contain additional Met-rich sequences inserted at the COOH-terminal portion of the M-domain. SRP54sp contains a 480-nucleotide intron located 78 nucleotides from the 5' end of the open reading frame. Although the function of the yeast homologues is unknown, gene disruption experiments in S. cerevisiae show that the gene is essential for growth. The identification of SRP54sc and SRP54sp provides the first evidence for SRP related proteins in yeast.

Isolation and characterization of a laminin-binding protein from rat and chick muscle.

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.

Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.