Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by activating KRAS mutations and TP53 alterations. TP53 missense mutations lose their wild-type tumor-suppressor function. Here, we studied whether p53 missense mutations have potential gain-of-function oncogenic roles and their impact on cancer-cell-intrinsic gene expression and the tumor immune microenvironment (TME) in PDAC. p53R172H established an immunosuppressive TME and impaired the efficacy of immune checkpoint inhibitors (ICIs) by regulating a distinct set of chemokines. Among these, tumor-specific reduction of Cxcl1, which encodes a chemoattractant for neutrophils, promoted T cell infiltration and decreased tumor growth. Mechanistically, p53R172H occupied the distal enhancers of Cxcl1 and amplified its expression. These enhancers were responsible for Cxcl1 expression and were essential for its immunosuppressive function. Nuclear factor κB (NF-κB) was a critical cofactor required for p53R172H occupancy at these enhancers. Thus, a common mutation in a tumor-suppressor transcription factor appropriates enhancers, thereby stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC.

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is an aggressive malignancy complicated by poor early diagnosis and a lack of response to traditional treatments. It is characterized by a desmoplastic stroma, a lack of infiltration and activation of T cells, and a low mutational burden. The genetic landscape of PDAC is defined by activating KRAS mutations (~90%) and p53 alterations (~70%), but the molecular switches perturbed by these genetic aberrations remain unclear. p53 missense mutations, unlike mutations resulting in the loss of p53, are considered to acquire tumor-supporting functions. But these novel functions remain uncharacterized. The ambiguity in the molecular mechanism of mutant-p53 is further exacerbated by the existence of various types of p53 mutations. The majority of p53 mutations are missense mutations in the DNA binding domain. The repertoire of transcription factors (TFs) it can interact with and the vast regulatory landscape of each TF—composed of gene promoters and distal enhancers—present obstacles in understanding the molecular mechanisms promoting PDAC. In this study, we examined how a common p53 missense mutation in PDAC plays a role in weakening the Immune checkpoint Inhibitors (ICIs) efficacy. Using cells derived from a genetically engineered mouse model (GEMM) of PDAC with activating KRAS mutation (KrasG12D/+) and a p53 missense mutation (p53R172H/-), we found that the PDAC tumorigenesis and resistance to ICIs are dependent on the mutant-p53. We used isogenic p53-null PDAC cells and the restoration of p53R172H in p53-null cells to demonstrate the role of p53R172H in controlling the expression of immunosuppressive chemokine genes such as Cxcl1. p53R172H deletion attenuated PDAC tumor growth, increased the influx of cytotoxic T-cells, and sensitized the tumor to ICIs. The p53R172H-mediated TME reprogramming was replicated by the deletion of the Cxcl1 gene, suggesting the anti-tumorigenic effect of p53R172H was mediated by the Cxcl1 gene. We probed the mechanism of Cxcl1 expression dependence on p53R172H. We found that in conjunction with NF-kB, p53R172H occupies the distal transcription regulatory elements (dTREs) of the Cxcl1 gene harboring NF-kB binding sites. Strikingly, deletion of the Cxcl1 dTREs in PDAC cells recapitulates the phenotypes of p53R172H deletion and Cxcl1 deletion in terms of tumor size, immune landscape of the TME, and ICI responsiveness. Furthermore, we examined the interplay between p53R172H and NF-kB and found that the p53R172H physically interacts with the NF-kB subunit RelA and facilitates its nuclear translocation. Overall, we characterize how a common p53 mutation in PDAC co-opts non-coding regulatory DNA to augment the expression of selective chemokine genes and establishes an immunosuppressive TME to shield the therapeutic benefits of ICIs. Citation Format: Dig B. Mahat, Heena Kumra, Emily Metcalf, Sarah Castro, Kim Nguyen1, Arundeep Singh, William W. Ho, Ivy Chen, Brandon Sullivan, Leon Yim, Enrico Moiso, Vikash Chauhan, Hernandez Moura Silva, Stefani Spranger, Rakesh Jain, Phillip A. Sharp. Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B087.

Background Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, M1 macrophages are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 to treat hepatocellular carcinoma. The CAR serves as a homing ‘GPS’ signal for trafficking directly to the tumor site, and directs phagocytosis specifically at targeted tumor cells. Our proprietary M83.CAR molecule contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83.CAR-M cells are not inhibited by the prevalent CD47 ‘do not eat me’ checkpoint, which is known to restrict myeloid function in the TME. Methods Primary hematopoietic stem cells (HSCs) were harvested and engineered to express a CAR molecule by lentivirus transduction generating CAR-HSCs. The CAR-HSCs underwent our proprietary ex-vivo HSC differentiation process to yield CAR-Monocytes. Results Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages enables robust tumor cell phagocytosis, the central mechanism that results in the following: 1) proinflammatory shift in the TME, 2) recruitment of APCs and immune cells, 3) activation of T-cells against tumor neoantigens. Conclusions The above summarizes a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Our proprietary HSC-derived CAR-M platform yields a unique product that demonstrates durability and superior function, which we expect to translate to the clinic.

'Candidatus Liberibacter solanacearum' (Lso) is a bacterial pathogen that causes 'zebra chip' disease in potato. Vectored by the potato psyllid Bactericera cockerelli, Lso inflicts severe economic losses annually in solanaceous and other crops. Conventional management strategies have been insufficient to control the pathogen. As an environmentally friendly phytosanitary technique, electron beam (eBeam) irradiation has been shown to drastically suppress psyllid growth and development. However, whether this chemical-free technology negatively impacts the pathogen transmission remains to be investigated. In this study, we irradiated newly emerged adult psyllids and measured abundance of Lso they carried. An eBeam dose of 100 Gy significantly suppressed Lso in irradiated psyllids, although a rebound was detected on day 7 post irradiation. This resumed bacterial growth was not observed in psyllids irradiated at 250 or 500 Gy. Electrical penetration graphs revealed that eBeam substantially impacted psyllid feeding activity. No significant difference was detected in actin cytoskeleton structure and nucleus integrity of its midgut cells 1 or 7 days post eBeam irradiation at 500 Gy. Furthermore, infection of tomato plants was drastically reduced when fed upon by psyllids exposed to 250 Gy and undetectable at 500 Gy. Thus, eBeam significantly lowered psyllid's transmission efficiency and even abolished this capacity. Taken together, eBeam irradiation curtailed Lso titers in potato psyllids and impaired its bacterial transmission. Our results have demonstrated the real potential of irradiation technology in management of insect-vectored diseases.

Supplementary Tables S1 - S6. Supplementary Table S1. CT values for demosplasia-related genes in PAN02 tumors. Data obtained from PCR array. Supplementary Table S2. CT values for demosplasia-related genes in AK4.4 tumors. Data obtained from PCR array. Supplementary Table S3. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S4. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) {less than or equal to}25. Supplementary Table S5. Univariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25. Supplementary Table S6. Multivariate analysis of prognostic factors for resected pancreatic cancer patients with body mass index (BMI) >25.

Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glio-blastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.

All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they cannot synthesize sterols de novo and must acquire sterols through diet. Cholesterol is the dominant sterol in most insects; however, because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. An additional challenge for plant-feeding insects is that only limited types of phytosterols can be converted to cholesterol. In this study, we used RNA interference to modify the plant sterol profile to generate phytosterols that would negatively impact insect herbivores. Specifically, we knocked down the expression of Arabidopsis thaliana Δ8–Δ7-sterol isomerase gene (HYD1), responsible for the conversion of Δ8-sterols into Δ7-sterols. Silenced lines with > 95

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (FIt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immuno-therapies against pMMR CRC.

Abstract All eukaryotes use sterols as structural components in cell membranes and as precursors for key hormones. However, arthropods are unique among eukaryotes because they are unable to synthesize sterols de novo and must acquire sterols through their diet. Cholesterol is the dominant sterol in most insects, but because plants contain little cholesterol, most insect herbivores convert phytosterols to cholesterol. However, metabolic constraints limit which phytosterols insect herbivores can convert to cholesterol. In this study we used RNA interference to modify the sterol profile of Arabidopsis thaliana to generate phytosterols that would be problematic for insect herbivores. We did this by silencing the Δ8-Δ7-sterol isomerase gene (HYD1) that is responsible for the conversion of Δ8-sterols into Δ7-sterols. Sterol production decreased in Arabidopsis lines with > 95% transcript reduction and half of the total sterol profile in these plants showed a Δ8 configuration. These sterol-modified lines exhibited normal growth, but caterpillars (Plutella xylostella) reared on these plants showed reduced growth and survival, while adults showed decreased reproductive output. Sterol analysis of the pupae reared on sterol-modified plants suggests these negative effects are a function of reduced cholesterol levels and the accumulation of Δ8-sterols. Finally, we used our collective performance and reproduction data to model the effects of sterol-modified plants at the population level. We show that population numbers are significantly reduced, and generation time extended, after only two generations. Our results suggest that modifying plant sterols has real potential for management of pest caterpillars, especially multivoltine species.

Immune checkpoint blockers (ICBs) have revolutionized the treatment of some solid cancers but have failed to benefit the majority of glioblastoma (GBM) patients. Two reasons underlying limited ICB benefit are: 1) immune-related adverse events, and 2) resistance conferred by the tumor microenvironment. Here, we show that ICBs induce cerebral edema in patients and GBM mouse models. This edema results from an inflammatory response to ICB treatment that disrupts the blood-tumor-barrier, as confirmed by intravital imaging, mechanistic blocking studies, and single-cell RNA sequencing. Losartan – a commonly prescribed antihypertensive agent – controls ICB-induced edema, reprograms the immunosuppressive tumor microenvironment, and improves survival under ICB therapy. In combination with a standard of care regimen in mice mimicking clinical treatment (surgical resection, chemoradiation), losartan increases the percent of long-term surviving (cured) mice under ICB therapy from 16% to 43%. Finally, a bihemispheric “resect-and-response” model to establish predictive biomarkers from the tumor microenvironment reveals that cured mice have an immunostimulatory (“hot”) immune tumor compartment prior to therapy. These results provide the basis for clinical testing of adding to losartan to ICB treatment for GBM patients.

Oxygen (O2) plays an essential role in aerobic organisms including terrestrial insects. Under hypoxic stress, the cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth. However, larvae, particularly 4th instar larvae exhibit very high tolerance to hypoxia and can recover normal growth once brought to normoxia. To better understand the molecular mechanism that enables insects to cope with low O2 stress, we performed RNA-seq to distinguish hypoxia-responsive genes in midguts and subsequently identified potential common cis-elements in promoters of hypoxia-induced and -repressed genes, respectively. Selected elements were subjected to gel-shift and transient transfection assays to confirm their cis-regulatory function. Of these putative common cis-elements, AREB6 appeared to regulate the expression of CmLPCAT and CmScylla, two hypoxia-induced genes. CmZFH, the putative AREB6-binding protein, was hypoxia-inducible. Transient expression of CmZFH in Drosophila S2 cells activated CmLPCAT and CmScylla, and their induction was likely through interaction of CmZFH with AREB6. Binding to AREB6 was further confirmed by bacterially expressed CmZFH recombinant protein. Deletion analyses indicated that the N-terminal zinc-finger cluster of CmZFH was the key AREB6-binding domain. Through in silico and experimental exploration, we discovered novel transcriptional regulatory components associated with gene expression dynamics under hypoxia that facilitated insect survival.

Strong and durable anticancer immune responses are associated with the generation of activated cancer-specific T cells in the draining lymph nodes. However, cancer cells can colonize lymph nodes and drive tumour progression. Here, we show that lymphocytes fail to penetrate metastatic lesions in lymph nodes. In tissue from patients with breast, colon, and head and neck cancers, as well as in mice with spontaneously developing breast-cancer lymph-node metastases, we found that lymphocyte exclusion from nodal lesions is associated with the presence of solid stress caused by lesion growth, that solid stress induces reductions in the number of functional high endothelial venules in the nodes, and that relieving solid stress in the mice increased the presence of lymphocytes in lymph-node lesions by about 15-fold. Solid-stress-mediated impairment of lymphocyte infiltration into lymph-node metastases suggests a therapeutic route for overcoming T-cell exclusion during immunotherapy. In metastatic lymph nodes, solid stress generated by metastatic growth prevents the infiltration of lymphocytes into the lesions.

Significance Immune checkpoint blockade (ICB) has been efficacious in several cancer types. However, mismatch repair–proficient (pMMR) metastatic colorectal cancer (CRC), ∼95% of total metastatic CRC cases, typically does not respond to ICB. Here, we show that orthotopic liver metastasis mouse models of pMMR CRC cell lines are unresponsive to ICB and recapitulate the resistance of human disease, unlike subcutaneous tumors of the same cell lines. We also show that just like the human disease, orthotopic pMMR CRC liver metastases have a paucity of T cells and dendritic cells, and that treatment with Flt3L sensitizes the liver metastases to ICB. Our findings highlight that orthotopic tumor models, and not subcutaneous models, should be used for preclinical studies of cancer immunotherapy.

Background Patients with metastatic HER2/neu-positive (HER2/neu +) breast cancer (BC) often experience treatment resistance, disease recurrences and metastases. Thus, new approaches for improving the treatment of HER2/neu + BC to prevent metastatic dissemination are urgently needed. Our previous studies have shown that losartan, an angiotensin receptor blocker, increases tumor perfusion and decreases hypoxia in a number of tumor models. Hypoxia reduces the efficacy of radiation and increases metastases. We therefore hypothesized that by modifying tumor stroma and increasing oxygenation, losartan will improve the outcome of radiotherapy and inhibit disease progression in a highly metastatic HER2/neu + murine BC model. Methods We established a metastatic HER2/neu + murine BC line (MCa-M3C) and used it to generate mammary fat pad isografts in syngeneic female FVB/N mice. Starting on day 3 after orthotopic tumor implantation, we administered a 7-day losartan treatment (40 mg/kg BW, gavage daily); or a 7-day losartan treatment followed by 20 Gy single dose local irradiation (S-IR) on day 10 (tumor size ~ 100 mm 3 ), or 20 Gy local fractionated (5 × 4 Gy daily) irradiation (F-IR) on days 10–14. We analyzed tumor-growth delay (TGD), development of spontaneous lung metastases, animal survival, tumor vascular density, and tumor hypoxia. Results Treatments with S-IR, F-IR, Losartan + S-IR, or Losartan + F-IR resulted in a significantly increased TGD (8–16 days) in MCa-M3C tumors versus controls. However, the combination of Losartan + S-IR and Losartan + F-IR further enhanced tumor response to radiation alone by increasing TGD an additional 5 to 8 days for both single and fractionated dose irradiation ( P < 0.01), decreasing lung metastasis (Losartan + IR vs. Control, P < 0.025), and increasing animal survival (Losartan + IR vs. Control, P = 0.0303) . In addition, losartan treatment significantly increased tumor vascularity ( P = 0.0314) and decreased pimonidazole positive (hypoxic) area ( P = 0.0002). Conclusions Combining losartan with local irradiation significantly enhanced tumor response, at least in part via reduced tumor hypoxia presumably due to increased tumor perfusion. Our findings suggest that combining losartan with radiotherapy is a potential new treatment strategy for local control and inhibiting metastasis in HER2 + BC.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2-positive breast cancer and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, resulting in site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase reduces human epidermal growth factor receptor 2-positive breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies. Ferraro et al. report that fatty acid synthesis is needed for brain cancer metastasis and show that blocking this process by inhibiting fatty acid synthase reduces the metastatic growth of breast cancer cells in the brain.

Brain metastases are refractory to therapies that control systemic disease in patients with human epidermal growth factor receptor 2-positive breast cancer and the brain microenvironment contributes to this therapy resistance. Nutrient availability can vary across tissues, therefore metabolic adaptations required for brain metastatic breast cancer growth may introduce liabilities that can be exploited for therapy. Here we assessed how metabolism differs between breast tumors in brain versus extracranial sites and found that fatty acid synthesis is elevated in breast tumors growing in the brain. We determine that this phenotype is an adaptation to decreased lipid availability in the brain relative to other tissues, resulting in site-specific dependency on fatty acid synthesis for breast tumors growing at this site. Genetic or pharmacological inhibition of fatty acid synthase reduces human epidermal growth factor receptor 2-positive breast tumor growth in the brain, demonstrating that differences in nutrient availability across metastatic sites can result in targetable metabolic dependencies.

Abstract Purpose: A phase II trial in patients with locally-advanced pancreatic cancer (LAPC) revealed unprecedented rates of complete surgical resection after adding losartan (L) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) (NCT01821729). The aim of this study was to identify potential mechanisms of benefit by assessing the effects of FFX-L+CRT and FFX+CRT on the stromal tumor microenvironment. Experimental Design: We performed a gene analysis of RNA extracted from pancreatic cancer (PC) tissue sections (NanoString nCounter PanCancer Immune Profiling Panel of 730 genes) and immunohistochemistry using surgical samples from patients treated with FFX+CRT (N=15), FFX-L+CRT (N=17) or underwent surgery upfront, without any neoadjuvant therapy (N=9). Results: In comparison to untreated PC, we found 314 and 243 differentially expressed genes (DEGs, adjusted p-value < 0.05) in FFX-L+CRT and FFX+CRT, respectively, and 54 DEGs between FFX-L+CRT and FFX+CRT. PCs from both neoadjuvant FFX-L+CRT and FFX+CRT had increased expression of genes linked to blood vessel maturation (CDH5, THBS1), transvascular migration of leukocytes (JAM3, PECAM1, MCAM, ICAM2), T cell activation (CD6, ALCAM, NFATC1), cytolytic activity of NK cells and T cells (GZMA, GZMB, GZMH, KLRB1) and dendritic cell (DC) related genes (CD209, CD1C, IL3RA). The FOXP3 gene — encoding for a transcription factor that regulates the activity of CD4+ regulatory T cells (Tregs)— was down-regulated in FFX-L+CRT versus untreated samples. Direct comparison of FFX-L+CRT versus FFX+CRT showed increased expression of genes involved in lymphocyte activation (NFATC4, DPP4, STAT5B), and reduced expression of genes that regulate B cell activity (CD22, TRAF3, MS4A1) and CEACAM6, which promotes PC invasion. We also analyzed the correlation of each gene (n=730) with overall survival (OS). For patients treated with FFX-L+CRT, improved OS was negatively correlated with genes that promote invasion in PC (PBK), B cell development and signaling (SYK, BLNK), infiltration of monocytes and macrophages in tumors (CCR2), immune checkpoint (BTLA) and inhibit angiogenesis (TNFSF15). In patients treated with FFX+CRT, OS was positively correlated with genes that stimulate inflammation (IL32), T cell and DC activation (CD48), presentation of glycolipid antigens (CD1E) and antigen processing and presentation by DCs (CD209). Immunohistochemistry studies revealed significantly less residual disease and a higher infiltration of CD8+CD3+ T cells in FFX-L+CRT than FFX+CRT treated tumors. In addition, in patients treated with FFX-L+CRT we found significantly fewer Tregs in PC lesions with a complete/near complete versus poor/no response, confirming our transcriptomic findings. Conclusions: Our findings suggest that FFX-L+CRT can normalize the vasculature, and reduce invasion and the immunosuppressive effects of B cells, CCR2-positive macrophages and Tregs, and thus improve treatment outcome in patients with LAPC, although additional studies are needed. Citation Format: Yves Boucher, Jessica M. Posada, Sonu Subudhi, Ashwin S. Kumar, Ivy X. Chen, Mei R. Ng, Mari Mino-Kenudson, Nilesh Talele, Dan G. Duda, Dai Fukumura, Janet E. Murphy, Jeffrey W. Clark, David P. Ryan, Carlos Fernandez-Del Castillo, Theodore S. Hong, Rakesh K. Jain. Addition of losartan to FOLFIRINOX and chemoradiation reduces the expression of pro-invasive and immunosuppressive genes in locally-advanced pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-097.

BACKGROUND OverexpressingCIRCADIAN CLOCK ASSOCIATED1inArabidopsis thaliana(CCA1-ox) increases indole glucosinolate production and resistance to green peach aphid (Myzus persicae). Little is known of how aphids respond to this group of plant defense compounds or of the underlying molecular mechanism. Results Aphids reared onCCA1-oxfor over 40 generations (namely the CCA population) became less susceptible toCCA1-oxthan aphids maintained on the wild-type Col-0 (namely the COL population). This elevated tolerance was transgenerational as it remained for at least eight generations after the CCA population was transferred to Col-0. Intriguingly, transcriptome analysis indicated that all differential cytochrome P450 monooxygenase genes (MpCYPs), primarilyMpCYP4s,MpCYP380sandMpCYP6s, were more highly expressed in the CCA population. Application of a P450 inhibitor to the CCA population resulted in decreased aphid reproduction onCCA1-ox, which was not observed if aphids were reared on Col-0. When indole glucosinolate biosynthesis inCCA1-oxwas blocked using virus-induced gene silencing, the effect of the P450 inhibitor on the CCA population was attenuated, affirming the essential role played by MpCYPs in counteracting the defense mechanism inCCA1-oxthat is low or absent in Col-0. Furthermore, we used host-induced gene silencing to identify MpCYP380C6 and MpCYP380C9 that specifically facilitated the CCA population to cope withCCA1-mediated plant defense. Expression profiles revealed their possible contribution to the transgenerational tolerance observed in aphids. Conclusion MpCYP380C6 and MpCYP380C9 in aphids play a crucial role in mitigating indole glucosinolate-mediated plant defense, and this effect is transgenerational.

Objective Intrahepatic cholangiocarcinoma (ICC)—a rare liver malignancy with limited therapeutic options—is characterised by aggressive progression, desmoplasia and vascular abnormalities. The aim of this study was to determine the role of placental growth factor (PlGF) in ICC progression. Design We evaluated the expression of PlGF in specimens from ICC patients and assessed the therapeutic effect of genetic or pharmacologic inhibition of PlGF in orthotopically grafted ICC mouse models. We evaluated the impact of PlGF stimulation or blockade in ICC cells and cancer-associated fibroblasts (CAFs) using in vitro 3-D coculture systems. Results PlGF levels were elevated in human ICC stromal cells and circulating blood plasma and were associated with disease progression. Single-cell RNA sequencing showed that the major impact of PlGF blockade in mice was enrichment of quiescent CAFs, characterised by high gene transcription levels related to the Akt pathway, glycolysis and hypoxia signalling. PlGF blockade suppressed Akt phosphorylation and myofibroblast activation in ICC-derived CAFs. PlGF blockade also reduced desmoplasia and tissue stiffness, which resulted in reopening of collapsed tumour vessels and improved blood perfusion, while reducing ICC cell invasion. Moreover, PlGF blockade enhanced the efficacy of standard chemotherapy in mice-bearing ICC. Conclusion PlGF blockade leads to a reduction in intratumorous hypoxia and metastatic dissemination, enhanced chemotherapy sensitivity and increased survival in mice-bearing aggressive ICC.

Abstract Introduction: Only a fraction of hepatocellular carcinoma (HCC) patients benefit from programmed death receptor-1 (PD-1) blockade. Combining PD-1 blockade with antiangiogenic treatments has been able to substantially increase the fraction of responsive HCC patients, but the mechanism of this interaction is unknown. We recapitulated these clinical outcomes (response vs. resistance) using orthotopic murine models of HCC. VEGFR2 blockade significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 monotherapy conferred a minor survival. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in multiple models. Combination therapy reprogrammed the immune microenvironment by increasing CD8+ cytotoxic T-cell infiltration and activation, improving the ratio of anti- vs. pro-tumor-associated macrophages, and reducing T regulatory cell (Treg) infiltration in HCC tissue. Moreover, we found that VEGFR-2 blockade increased the PD-1 expression in tumor-infiltrating CD4+ cells. Under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy. Conclusion: We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase survival in both anti-PD-1 therapy resistant and responsive HCC models. Citation Format: Meenal Datta, Kohei Shigeta, Tai Hato, Shuji Kitihara, Ivy X. Chen, Aya Matsui, Hiroto Kikuchi, Emilie Mamessier, Shuichi Aoki, Rakesh R. Ramjiawan, Hiroki Ochiai, Nabeel Bardeesy, Peigen Huang, Rakesh K. Jain, Mark Cobbold, Andrew X. Zhu, Dan G. Duda. Dual PD-1 and VEGFR-2 blockade induces vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B89.