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Therapy failure and metastasis-associated mortality are stumbling blocks in the management of PDAC in patients. Failure of therapy is associated to intense hypoxic conditions of tumors. To develop effective therapies, a complete understanding of hypoxia-associated changes in genetic landscape of tumors during disease progression is needed. Because artificially immortalized cell lines do not rightly represent the disease progression, studying genetics of tumors in spontaneous models is warranted. In the current study, we generated a spectrum of spontaneous human (UM-PDC1; UM-PDC2) and murine (HI-PanL, HI-PancI, HI-PanM) models representing localized, invasive, and metastatic PDAC from a patient and transgenic mice (K-rasG12D/Pdxcre/Ink4a/p16-/). These spontaneous models grow vigorously under hypoxia and exhibit activated K-ras signaling, progressive loss of PTEN, and tumorigenicity in vivo. Whereas UM-PDC1 form localized tumors, the UM-PDC2 metastasize to lungs in mice. In an order of progression, these models exhibit genomic instability marked by gross chromosomal rearrangements, centrosome-number variations, Aurora-kinase/H2AX colocalization, loss of primary cilia, and α-tubulin acetylation. The RNA sequencing of hypoxic models followed by qRT-PCR validation and gene-set enrichment identified Intestine-Specific Homeobox factor (ISX)–driven molecular pathway as an indicator PDAC aggressivness. TCGA-PAAD clinical data analysis showed high ISX expression correlation to poor survival of PDAC patients, particularly women. The functional studies showed ISX as a regulator of i) invasiveness and migratory potential and ii) VEGF, MMP2, and NFκB activation in PDAC cells. We suggest that ISX is a potential druggable target and newly developed spontaneous cell models are valuable tools for studying mechanism and testing therapies for PDAC.

Histone H3.3 G34R/V mutations are drivers of pediatric high-grade glioma (pHGG). However, the mechanism(s) responsible for G34R/V induced tumorigenesis are unclear. We observed that pHGG cells with H3.3 G34 mutations have significantly reduced phosphorylation at H3.3 S31 as compared to H3.3 WT cell lines. And, in vitro, G34 mutation completely blocks H3.3 S31 phosphorylation by Chk1 – the kinase responsible for S31 phosphorylation. During early mitosis, phospho-S31 is enriched in the chromosomal pericentromere; which plays a vital structural role in chromosome segregation. Over-expression of GFP-H3.3 G34R or non-phosphorylatable GFP-H3.3 S31A in chromosomally stable cell lines results in a significant increase in chromosome mis-segregations. Likewise, H3.3 G34V pHGG derived cells showed a higher frequency of mis-segregation than H3.3 WT cells. During cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads to the chromosome arms where it stimulates p53 accumulation in G1 – thus acting as a sensor to suppress the proliferation of aneuploid cells. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. Taken together this work demonstrates that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we expressed WT H3.3, H3.3-G34R or H3.3-S31A in a mouse model of pHGG. H3.3 WT controls developed low-grade tumors and all survived 80 days. Mice with H3.3-G34R or H3.3-S31A developed low- and high-grade tumors. And 78% and 50% of H3.3-G34R and H3.3-S31A mice, respectively, survived to 80 days. Our work suggests that H3.3 G34R/V drives chromosomal instability through the suppression of H3.3 S31 phosphorylation AND that chromosomal instability is a contributing driver of glioma formation in G34 mutant tumors.

Adherent cells cultured on flat, homogeneous surfaces typically maintain an intact cell body with a polygonal or fan shape, despite active migration and strong mechanical interactions with the substratum. We hypothesized that, in addition to the constraint of the surface membrane, an active mechanism may be involved in maintaining the shape and integrity of the cell body particularly where cells encounter complex topographic patterns of guidance cues. To detect if there is a mechanism that constrains cell shape, we plated NIH 3T3 fibroblasts on ring-patterned substrata 8–17.5 microns in width and 53–133 microns in outer diameter. Untreated cells have a limited angular span, encompassing an average of 108 degrees around the ring, even though these cells were able to cover a much larger surface when plated on flat surfaces of the same material. Treatment of 3T3 cells with a myosin II inhibitor, blebbistatin, induced a striking increase in the bending ability, causing cells to cover more than 60% of the ring. Inhibition of the Rho-dependent kinase with Y-27632 caused a similar but smaller increase in the angular span. Our results suggest that cell shape is controlled not only by the passive constraint of the surface membrane but also by an active mechanism driven by myosin II-mediated contractility under the regulation of Rho-dependent kinase. The inward surface tension-like forces allow the cell to maintain its integrity while navigating through complex physiological environments.

The study of mitosis has always relied on bulk-preparation biochemistry techniques ( Mazia & Dan, 1952 ), but very early on lent itself to living, single cell microscopic techniques ( Inoue, 1953 ; Taylor, 1959 ). Here we describe several of the methods used by our lab to study cell division in living cultured cells, including cold-induced mitotic arrest, cold-induced chromosome missegregation, same-cell live and fixed cell imaging, and microinjection of inactivating antibodies. We detail our imaging system based on an upright fluorescent microscope and spinning disk confocal, as well as the customized “HEKS” metal support slide imaging chambers.

BACKGROUND: Heterozygous mutations in Histone H3.3 (K27M, G34R/V) occur in 70–80% of pediatric high-grade gliomas and 78% of diffuse intrinsic pontine gliomas (DIPG). These mutations are also found in low-grade gliomas that progress to higher grade tumors. Epigenetic dysregulation resulting from altered methylation at or near the mutated sites is believed to drive malignant transformation H3 differs from H3.1 by only 5 amino acids, which includes a unique phosphorylation site, Serine 31. S31 is phosphorylated by Chk1 kinase at pericentromeric heterochromatin in mitosis and on the arms of missegregated chromosomes. Masking S31 phosphorylation by antibody injection or using a non-phosphorylatable S31A substitution prevents cell cycle arrest in response to chromosome missegregation. OBJECTIVE: To determine if H3.3 K27M or G34R/V mutations alter the regulation of S31 phosphorylation to promote chromosome missegregation, aneuploidy, and tumorigenesis. METHODS: Live cell imaging and an established mouse model of glioma. RESULTS: DIPG cells with the K27M mutations show decreased mitotic Ser31 phosphorylation and increased chromosome missegregation. In both model cell lines and normal astrocytes, K27M and G34R mutations abolish S31 phosphorylation. Expression of S31A, K27M, or G34R mutants results in increased chromosome missegregation and aneuploidy, whereas cells expressing phosphomimetic double mutants (K27M/S31E or G34R/S31E) divide normally. Using the RCAS/TVA mouse model, H3.3WT, K27M, and S31A constructs were introduced into the brains of newborn mice along with mouse BRAF(V600E). 0/13 tumors developed in the BRAF(V600E) + H3.3WT cohort; however, 80% (16/20) of H3.3K27M + BRAF(V600E) and 70% (7/10) H3.3S31A + BRAF(V600E) cohorts developed high-grade gliomas within 100 days. CONCLUSION: A single heterozygous H3.3 mutation is sufficient to generate aneuploid cells, allows them to reenter the cell cycle and to promote tumorigenesis. Restoring cell cycle checkpoint activity by modulating S31 phosphorylation may be a rational treatment for DIPG or to prevent the progression of low-grade gliomas with H3.3 mutations.

The histone H3.3 G34R/V mutations are known drivers of high-grade pediatric glioma (pHGG). However, the mechanism(s) for H3.3 G34R/V induced tumor formation are unclear. Chk1 phosphorylates H3.3 S31 at the pericentromere during early mitosis, suggesting a novel mitotic function. We observed that H3.3 G34 mutant pHGG cells have reduced mitotic H3.3 S31 phosphorylation compare to WT H3.3 cell lines. The H3.3 G34R mutation reduced Chk1 phosphorylation at S31 by >90% in an in vitro kinase assay. Overexpression of either H3.3 G34R or non-phosphorylatable S31A in H3.3 WT, diploid cells led to a significant increase in chromosome mis-segregations. Likewise, H3.3 G34 mutant pHGG cells have significantly elevated rates of mis-segregation as compare to H3.3 WT pHGG cells. During normal cell division, phospho-S31 is lost in late anaphase. However, when chromosome missegregation occurs, phospho-S31 spreads and stimulates p53 accumulation in G1 – thus suppressing aneuploid cell proliferation. Here we show that cells expressing mutant G34 fail to arrest following mis-segregation, despite having WT p53. These studies demonstrate that the H3.3 G34R/V mutations are sufficient to transform normal, diploid cells into proliferative, chromosomally instable cells. To determine if this process contributes to tumorigenesis, we used the RCAS/TVA mouse model to overexpress H3.3 WT, G34R, or S31A in the glial precursor cells of mice pups. Over 100 days, S31A and G34R mice had drastically reduced survival (averaging 77, 81, and 100 days for S31A, G34R, and WT mice). Furthermore, most G34R and S31A mice developed HGG, while H3.3 WT mice remained tumor-free and did not develop high-grade tumors. Our work strongly indicates that a major factor in H3.3 G34R pHGG formation is the induction of chromosomal instability – which occurs directly through the suppression of H3.3 S31 phosphorylation.

Diffuse midline glioma (DMG) is a lethal brain tumor that typically occurs in children. Numerous studies have demonstrated the central role of the H3K27M mutation and secondary loss of H3K27 trimethylation (H3K27me3) in DMG tumorigenesis. Understanding how the H3K27M mutation alters the epigenetic landscape of the cell is necessary for revealing molecular targets that are critical to tumorigenesis. To investigate the epigenetic effects of H3K27M mutation in DMG, we developed revertant DMG cell lines with the mutant methionine residue reverted to wildtype (i.e., M27K). Revertant cells were analyzed for epigenetic changes and phenotypic differences in vitro and in vivo. H3M27K DMG cells grew in culture but displayed diminished proliferative capacity. H3M27K cells demonstrated total loss of H3K27M expression and restored trimethylation of H3K27 and H3K4. Furthermore, consistent with the hypothesis that the H3K27M mutation impacts H3 phosphorylation via expression of Aurora Kinase during mitosis, H3M27K cells demonstrated reduced expression of both Aurora Kinase A and phosphorylation of H3 serine residues 10 and 28. In line with the critical role of H3S10 phosphorylation in chromatin segregation, H3M27K cells also demonstrated restored chromosome segregation compared to H3K27M cells. In vivo data will be discussed. Revertance of the H3K27M mutation reduces tumorigenesis in DMG tumors. Isogenic H3M27K cells display reversal of key epigenetic changes associated with oncogenesis in DMG. The revertant H3M27K DMG model is a useful tool to investigate the downstream epigenetic reprogramming specific to H3K27M mutation in these tumors.

Glycolipid transfer proteins (GLTPs) were first identified over three decades ago as ~24kDa, soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. Upon discovery that GLTPs use a unique, all-α-helical, two-layer ‘sandwich’ architecture (GLTP-fold) to bind glycosphingolipids (GSLs), a new protein superfamily was born. Structure/function studies have provided exquisite insights defining features responsible for lipid headgroup selectivity and hydrophobic ‘pocket’ adaptability for accommodating hydrocarbon chains of differing length and unsaturation. In humans, evolutionarily-modified GLTP-folds have been identified with altered sphingolipid specificity, e. g. ceramide-1-phosphate transfer protein (CPTP), phosphatidylinositol 4-phosphate adaptor protein-2 (FAPP2) which harbors a GLTP-domain and GLTPD2. Despite the wealth of structural data (> 40 Protein Data Bank deposits), insights into the in vivo functional roles of GLTP superfamily members have emerged slowly. In this review, recent advances are presented and discussed implicating human GLTP superfamily members as important regulators of: i) pro-inflammatory eicosanoid production associated with Group-IV cytoplasmic phospholipase A(2); ii) autophagy and inflammasome assembly that drive surveillance cell release of interleukin-1β and interleukin-18 inflammatory cytokines; iii) cell cycle arrest and necroptosis induction in certain colon cancer cell lines. The effects exerted by GLTP superfamily members appear linked to their ability to regulate sphingolipid homeostasis by acting in either transporter and/or sensor capacities. These timely findings are opening new avenues for future cross-disciplinary, translational medical research involving GLTP-fold proteins in human health and disease. Such avenues include targeted regulation of specific GLTP superfamily members to alter sphingolipid levels as a therapeutic means for combating viral infection, neurodegenerative conditions and circumventing chemo-resistance during cancer treatment.

The macroautophagy/autophagy and inflammasome pathways are linked through their roles in innate immunity and chronic inflammatory disease. Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates pro-inflammatory eicosanoid production. Whether C1P also regulates autophagy and inflammasome assembly/activation is not known. Here we show that CPTP (a protein that traffics C1P from its site of phosphorylation in the trans-Golgi to target membranes) regulates both autophagy and inflammasome activation. In human epithelial cells, knockdown of CPTP (but not GLTP [glycolipid transfer protein]) or expression of C1P binding-site point mutants, stimulated an 8- to 10-fold increase in autophagosomes and altered endogenous LC3-II and SQSTM1/p62 protein expression levels. CPTP depletion-induced autophagy elevated early markers of autophagosome formation (Golgi-derived ATG9A-vesicles, WIPI1), required key phagophore assembly and elongation factors (ATG5, ATG7, ULK1), and suppressed MTOR phosphorylation and that of its downstream target, RPS6KB1/p70S6K. Wild-type CPTP overexpression exerted a protective effect against starvation-induced autophagy. In THP-1 macrophage-like surveillance cells, CPTP knockdown induced not only autophagy but also elevated CASP1/caspase-1 levels, and strongly increased IL1B/interleukin-1β and IL18 release via a NLRP3 (but not NLRC4) inflammasome-based mechanism, while only moderately increasing inflammatory (pyroptotic) cell death. Inflammasome assembly and activation stimulated by CPTP depletion were autophagy dependent. Elevation of intracellular C1P by exogenous C1P treatment (instead of CPTP inhibition) also induced autophagy and IL1B release. Our findings identify human CPTP as an endogenous regulator of early-stage autophagosome assembly and inflammasome-driven, pro-inflammatory cytokine generation and release.

Diffuse intrinsic pontine glioma (DIPG) carries a signature mutation in histone H3.3 (K27M) that induces epigenetic reprogramming via suppressed Lys27 triple methylation (K27me3). H3.3 has a Ser at position 31, which is adjacent to the K27M DIPG mutation. H3.3 Ser31 is phosphorylated beginning in prophase. This phosphorylation is restricted to pericentromeric heterochromatin and becomes dephosphorylated in anaphase. Surprisingly, chromosome missegregation triggers Ser31 hyperphosphorylation on the lagging chromosome, and masking phosphoS31 prevents p53 accumulation; part of the G1 checkpoint response to aneuploidy. Whether the K27M mutation influences Ser31 phosphorylation, chromosome segregation or G1 checkpoint control is unknown. We show that K27M DIPG cells have reduced pericentromeric phosphoS31 and increased chromosome instability (CIN) compared to normal, diploid human cells. CRISPR-editing of K27M to M27K restored phosphoS31 to WT levels and dramatically decreased the rate of CIN. We also demonstrate that Chk1 is the H3.3 Ser31 kinase. S31A abolishes Ser31 phosphorylation by Chk1, while K27M reduces it by ~60% in vitro. Chk1 knockdown abolishes phosphoS31 in normal, diploid mitotic cells, and induces CIN. In normal, diploid cells, expression of S31A or K27M mutations increased chromosome missegregation, whereas cells expressing a phosphomimetic double mutant (K27M/S31E) divide normally. WT cells arrest following chromosome missegregation. Yet, normal cells expressing H3.3 K27M or S31A do not arrest - despite having wild-type p53. Finally, using RCAS/TVA, we expressed H3F3AS31A in the brains of BRAFV600E mice, and ~50% developed tumors, suggesting that loss of phosphoS31 alone is oncogenic. Importantly, H3.3 S31A-expressing cells are WT for K27me3. Our results demonstrate that expressing H3.3 K27M reduces the population of H3.3 that is phosphorylatable at Ser31, leading to chromosome missegregation and preventing G1 arrest – thus creating proliferating CIN cells. We propose that a single amino acid substitution drives oncogenesis of DIPG, at least impart, by triggering the evolution of complex karyotypes.

Introduction: Adeno-associated virus (AAV) is a small virus of the parvovirus family and is widely used in clinical-stage gene therapy because of its low toxicity and limited side effects. There are a number of naturally occurring serotypes with different tissue tropism and further engineering of the viral capsid can increase the vectors suitability for use as a cancer vaccine. We recently showed that AAV serotype 6 (AAV6) successfully delivers a transgene to dendritic cells (DC) in vitro. In the present study we analyze several rationally designed AAV6 mutants to more efficiently target DC and further increase an antigen-specific immune response. Methods: AAV6 mutants encoding ovalbumin, tyrosinase, or GFP were developed and produced in the lab by standard procedure. Vaccination was performed as a single intramuscular (i.m.) injection in C57BL/6 mice. The immune response was measured against both ovalbumin (Ova) as a model antigen and tyrosinase (Tyr) as an endogenous antigen. The Ova-specific CD8 T-cells were identified in peripheral blood and splenocytes by binding epitope-specific tetramers. Activity of antigen-specific CD8 cells was estimated by IFNy production measured by ELISpot, and by in vivo and in vitro killing assays. A humoral response was analyzed by the presence of antigen-specific antibodies in mouse serum measured by ELISA or by Western Blotting. Vaccination with GFP was used to show direct antigen expression in DC. Briefly, 3 days after i.m. injection DC from drained lymph nodes were isolated by FACS (CD11c+/MHC II+) and the presence of GFP expression was analyzed by wide-field fluorescence microscopy. Results: Several significant observations were made: (i) optimized AAV6 directly targets DC in vivo as evidenced by the presence of GFP positive DC in mouse drained lymph nodes after i.m. injection with AAV6-GFP; (ii) AAV6 vectors induced a strong immune response not only against a strong immunogen such as Ova but also against a weak endogenous antigen such as Tyr. (iii) Transgene-specific cytotoxic CD8 T- cells showed superior killing capabilities against cancer cells in vitro and in cytotoxic assay in vivo. (iv) AAV6 based vaccine generates high transgene-specific antibodies titers detectable starting 3 weeks after injection. Our data allows us to choose the AAV6 mutant with superior capacity to induce an immune response against a transgene. As a future direction we will optimize the expression cassette to directly rout an antigen into the MHC class II processing and presentation pathway which is known to dramatically increase vaccine potency. The combination of optimized AAV capsid and expression cassette will be used to confirm an ability of our vaccine approach to reduce tumor growth and extend survival in a murine tumor model. In addition, our technology is highly compatible with, and complementary to current cancer treatments, including other immunotherapy approaches such as checkpoint (PD-1/PD-L1) inhibitors. Citation Format: Karina Krotova, Andrew Day, Edward Hinchcliffe, George Aslanidi. Bioengineering AAV6 vector-based vaccine for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1467.

The synthetic Vitamin A analog fenretinide is a promising chemotherapeutic agent. In the current paper, the role of PKC was examined in fenretinide-induced apoptosis in lymphoid leukemia cells. Levels of proapoptotic cleaved PKC positively correlated with drug sensitivity. Fenretinide promoted reactive oxygen species (ROS) generation. The antioxidant Vitamin C prevented fenretinide-induced PKC cleavage and protected cells from fenretinide. Suppression of PKC expression by shRNA sensitized cells to fenretinide-induced apoptosis possibly by a mechanism involving ROS production. A previous study demonstrated that fenretinide promotes degradation of antiapoptotic MCL-1 in ALL cells via JNK. Now we have found that fenretinide-induced MCL-1 degradation may involve PKC as cleavage of the kinase correlated with loss of MCL-1 even in cells when JNK was not activated. These results suggest that PKC may play a complex role in fenretinide-induced apoptosis and may be targeted in antileukemia strategies that utilize fenretinide.

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

Ceramide 1-Phosphate (C1P) is a biologically active sphingolipid that stimulates cell proliferation, macrophage migration, and inflammatory events. We recently characterized a novel human lipid transfer protein encoded by GLTPD1 on chromosome 1 (locus 1p36.33). We named the protein, ceramide 1-phosphate transfer protein (CPTP) due to its lipid specificity and discovered that CPTP transports C1P to the plasma membrane from TGN after synthesis by ceramide kinase. CPTP depletion by RNAi leads to C1P accumulation in TGN (~6-fold), stimulating cPLA2α and inducing pro-inflammatory eicosanoid production (Simanshu et al., Nature 500: 463-67, 2013). Here, we investigated whether CPTP depletion leads to programmed cell death and discovered an autophagic response in human cervical cancer cell lines (HeLa) and human lung adenocarcinoma epithelial cells (A549). Transfection with ShRNA (pSuper.GFP.ShRNA.CPTP vector) resulted in GFP-LC3 puncta and acidophilic acridine orange staining by epifluorescence microscopy and be...

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.