Pham, Linh

Introduction: ASXL1-mutant Chronic Myelomonocytic Leukemia (CMML) is a high-risk myeloid neoplasm characterized by epigenetic remodeling and overexpression of leukemogenic driver genes. DLK1 (delta-like homolog 1) is a non-canonical Notch ligand, crucial for the proliferation of early hematopoietic progenitor cells and its expression is controlled by maternal imprinting. DLK1 expression is restricted to select progenitor populations, but was found to be overexpressed in several malignancies including ASXL1-mutant CMML. Here we investigate the molecular mechanisms that drive the aberrant re-expression of DLK1 and postulate potential targeted therapeutic interventions. Methods: We performed bulk RNA- and ChIP-seq (H3K4me1/3, H3K27ac/me3, H2AK119ub, H2BK20ac) on bone marrow mononuclear cells from 20 patients with CMML (11 ASXL1-mutant, 9 -wildtype). All libraries were sequenced on an Illumina NovaSeq instrument. Differential gene expression and histone modification occupancy between ASXL1-mutant and -wildtype patients were assessed. Single-cell RNA-seq was performed for a subset of patients to investigate the cell populations of interest for DLK1 expression. To assess DLK1 allelic expression, we identified a single nucleotide polymorphism (SNP) in the coding region of DLK1 (rs1802710) and used cases which were confirmed heterozygous by Sanger sequencing. DLK1 transcript levels were quantified by RT-PCR, and the relative abundance of alleles was measured using digital droplet PCR. We designed primers to amplify the region around our SNP of interest and utilized high mismatch discrimination locked nucleic acid (LNA) probes labeled with FAM (reference SNP allele) and HEX (alternate SNP allele) to quantify specific alleles. The number of RNA molecules originating from each allele was quantified by counting the number of droplets that fluoresced with the corresponding probe colors. Illumina 850K EPIC microarrays were used to evaluate DNA methylation. Drug studies were carried out in progenitor colony forming assays derived from ASXL1-mutant patients (bone marrow mononuclear cells) with the dual BRD4 and p300 / CBP inhibitor EP31670 (62 nM, 24-hour exposure), quantifying DLK1 expression by real-time RT-PCR. Results: This study included 20 patients with CMML (median age at diagnosis 74 years). All ASXL1 mutations were typical hotspot mutations (most commonly Gly646Trpfs*12) and the variant allele frequencies were compatible with heterozygosity (median 0.41). The spectrum of co-mutations was typical and included TET2 (23%), SRSF2 (16%), CBL (7%), among others. DLK1 was strongly overexpressed in ASXL1-mutant patients (log2 fold-change +4.0, FDR < 0.001). Allele-specific gene expression studies (n = 3) revealed the re-expression of the imprinted maternal DLK1 allele in ASXL1-mutant patients. Single-cell gene expression studies (n = 5) demonstrated that the DLK1 overexpression is restricted to the hematopoietic progenitor cell population. Differential methylation analysis between patients with ASXL1-mutant clonal hematopoiesis (n = 4) and matched controls (n = 8) identified two hypermethylated CpG islands in the DLK1 promoter region (cg18122767, Δß = +8.6%, p = 0.021 and cg15831296, Δß = +5.8%, p = 0.008). Histone modification analysis identified DLK1 as a p300 target gene by mapping H2BK20ac onto established ASXL1-mutant-specific enhancer regions (H3K4me1 + H3K27ac co-occupancy). The distal enhancer EH38D2760182 was predicted to bind p300 and to govern the expression of DLK1. ChIP-seq experiment in K-562 and Kasumi-1 cells showed the binding of BRD4 and p300 to EH38D2760182, respectively. Targeting BRD4 and p300 using EP31670 led to the downregulation of DLK1 expression (mean fold-change = 0.569, p < 0.001, n = 3) in progenitor colony forming assays. Conclusions: We observed the aberrant re-expression of the maternally imprinted DLK1 allele in patients with ASXL1-mutant CMML. Both DLK1 promoter hypermethylation and the genotype-specific activation of enhancers by p300 were identified as putative regulatory mechanisms. The distal enhancer EH38D2760182 was found to bind BRD4 and p300 and the pharmacologic disruption of BRD4 / p300 activity in model systems led to the downregulation of DLK1. The clinical-grade dual inhibitor of BRD and CBP / p300 EP31670 is currently being investigated in patients with ASXL1-mutant chronic myeloid neoplasms (NCT05488548).

Although CD19-directed chimeric antigen receptor T cell (CART19) therapy is highly effective and was FDA approved for certain B-cell malignancies, most patients relapse after CART infusion within the first 1-2 years due to inadequate CART expansion in vivo. Vesicular stomatitis virus (VSV) has the ability to infect and lyse cancer cells. Clinical trials of VSV oncolytic therapy indicate that VSV efficiently infects cancer cells as well as innate immune cells. Therefore, we hypothesized that in patients who achieve suboptimal response to CART19, VSV engineered to express CD19 will augment anti-tumor activity through 1) direct lysis of cancer cells and 2) infecting cancer cells and innate immune cells with CD19 to further stimulate CART19. To test our hypothesis, human CD19 or GFP (control) was engineered between the glycoprotein and large-protein (Fig.1A) in a modified VSV backbone. A matrix inactivating mutation (M51R) rendered it incapable of suppressing anti-viral reactions of infected targets, potentially promoting its immunogenicity. First, we tested the anti-tumor activity of VSV-CD19 and VSV-GFP against the luciferase (luc)+CD19+ acute lymphoblastic leukemia cell line NALM6 and the luc+CD19- acute myeloid leukemia cell line MOLM13. VSV-CD19 and VSV-GFP successfully lysed NALM6 (Fig.1B) or MOLM13, both in vitro and in vivo (data not shown). Next, we investigated the efficiency of VSV-CD19 in infecting tumor and immune cells. 24 hours after exposure to VSV-CD19 or VSV-GFP, we analyzed the surface expression of CD19 on MOLM13 and revealed efficient CD19 delivery (Fig.1C). Next, we assessed VSV infection of peripheral blood mononuclear cells (PBMCs) from healthy donors (HDs). Freshly isolated HD PBMCs were infected with VSV-CD19 for 6 hours and subsequently assessed for CD19 expression. Consistent with findings from clinical trials, VSV-CD19 selectively infected and induced CD19 expression on monocytes while other cells were not affected (Fig.1D). To exclude potential toxicities against CART19, we co-cultured CART19 with VSV-CD19 or VSV-GFP using second-generation 4-1BB costimulated CART19. Both VSV-CD19 and VSV-GFP did not infect CART19 as evident by preservation of CART19 viability and lack of CD19 or GFP expression (Fig.1E). Having demonstrated that VSV-CD19 specifically delivered CD19 to monocytes, we next tested whether the infected monocytes stimulated CART19. VSV-CD19 infected monocytes induced potent antigen-specific proliferation of CART19 (Fig.1F) and resulted in enhanced anti-tumor activity against luc+NALM6 in vitro (Fig.1G). Next, we aimed to confirm these findings in vivo. We generated luc+CART19 to track CART19 expansion in vivo. Freshly isolated HD monocytes were infected with VSV-CD19 ex vivo. After 4 hours, VSV-CD19 was washed away and immunocompromised NSG mice were intravenously injected with VSV-CD19 infectedmonocytes. After 24 hours, 3.5x106 of luc+untransduced T cells (UTD) or luc+CART19 were injected intravenously. The T cell expansion was assessed by bioluminescence imaging (BLI). VSV-CD19 infected monocytes specifically stimulated and expanded CART19 (Fig.1H). Finally, we tested whether VSV-CD19 can stimulate and rescue suboptimal anti-tumor effects of CART19 in vivo using a NALM6 relapsed model. Here, 1x106 luc+NALM6 were injected intravenously into NSG mice on day -6. At day -1, mice were imaged and randomized according to tumor burden to receive 1x106 UTD or CART19 on day 0. Subsequently, at day 4, mice were re-imaged and randomized. At day 5, HD monocytes were injected intravenously. Three hours after administering monocytes, mice received 1x107 VSV-CD19 or VSV-GFP (Fig.1I). BLI revealed that CART19 plusVSV-CD19 showed better tumor control than CART19 monotherapy or CART19 plus VSV-GFP (Fig.1J-K). Furthermore, CART19 plus VSV-CD19 exhibited long-term survival (Fig.1L). In summary, VSV-CD19 not only demonstrated direct anti-tumor effects but also specifically delivered CD19 to monocytes and tumor cells, thereby re-stimulating and enhancing the anti-tumor activity of CART19. This work provides a rationale to study VSV-CD19 in patients who demonstrate only suboptimal response to CART19. This approach could also be applied to augment CART therapy in other tumors. Figure 1 Disclosures Sakemura: Humanigen: Patents & Royalties. Eckert:Genentech: Current Employment. Cox:Humanigen: Patents & Royalties. Parikh:Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; Verastem Oncology: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Merck: Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding. Kay:Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding. Peng:Imanis: Other: Equity Ownership. Russell:Imanis: Other: Equity Ownership. Kenderian:Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Lentigen: Research Funding; Torque: Consultancy; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding.

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.

While previous work has demonstrated that monosaccharides can be absorbed from bile, studies of sugar transport by the biliary, epithelia (i.e., cholangiocytes) are lacking. Using a novel model of polarized rat cholangiocytes in primary culture, designated normal rat cholangiocytes (NRC), we examined directly the uptake and transcellular transport of a nonmetabolizable monosaccharide, methyl alpha-D-glucopyranoside (AMG). When the apical or basolateral domain of cholangiocytes was exposed to radiolabeled AMG or sucrose (control), only apical absorption of AMG was evident. This apical uptake was time dependent, saturable, and significantly inhibited (> or = 90%) by removal of Na+ or in the presence of phlorizin (0.1 mM), a competitive inhibitor of the Na(+)-glucose cotransporter. The transcellular flux of AMG was also polar (i.e., apical to basolateral). Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of the transcript for the specific Na(+)-glucose cotransporter SGLT1 in NRC and in freshly isolated cholangiocytes but not in purified hepatocytes; in contrast, the transcript for SGLT2 was absent in all liver samples. In situ RT-PCR on frozen sections of normal rat liver showed that SGLT1 was expressed exclusively in cholangiocytes. Immunoblot analysis using a specific polyclonal antibody for the facilitative glucose transporter GLUT1 demonstrated it to be present in vesicles derived from NRC enriched in basolateral plasma membrane domains. Our data are consistent with the concept that SGLT1 is present on the apical domain of biliary epithelia and, in conjunction with GLUT1 on the basolateral domain, accounts for glucose absorption from bile.

Breast cancer radiotherapy increases the risk of heart failure with preserved ejection fraction (HFpEF). Cardiomyocytes are highly radioresistant, but radiation specifically affects coronary microvascular endothelial cells, with subsequent microvascular inflammation and rarefaction. The effects of radiation on left ventricular (LV) diastolic function are poorly characterized. We hypothesized that cardiac radiation exposure may result in diastolic dysfunction without reduced EF. Global cardiac expression of the sodium-iodide symporter (NIS) was induced by cardiotropic gene (adeno-associated virus serotype 9) delivery to 5-wk-old rats. SPECT/CT (125I) measurement of cardiac iodine uptake allowed calculation of the 131I doses needed to deliver 10- or 20-Gy cardiac radiation at 10 wk of age. Radiated (Rad; 10 or 20 Gy) and control rats were studied at 30 wk of age. Body weight, blood pressure, and heart rate were similar in control and Rad rats. Compared with control rats, Rad rats had impaired exercise capacity, increased LV diastolic stiffness, impaired LV relaxation, and elevated filling pressures but similar LV volume, EF, end-systolic elastance, preload recruitable stroke work, and peak +dP/d t. Pathology revealed reduced microvascular density, mild concentric cardiomyocyte hypertrophy, and increased LV fibrosis in Rad rats compared with control rats. In the Rad myocardium, oxidative stress was increased and in vivo PKG activity was decreased. Experimental cardiac radiation exposure resulted in diastolic dysfunction without reduced EF. These data provide insight into the association between cardiac radiation exposure and HFpEF risk and lend further support for the importance of inflammation-related coronary microvascular compromise in HFpEF. NEW & NOTEWORTHY Cardiac radiation exposure during radiotherapy increases the risk of heart failure with preserved ejection fraction. In a novel rodent model, cardiac radiation exposure resulted in coronary microvascular rarefaction, oxidative stress, impaired PKG signaling, myocardial fibrosis, mild cardiomyocyte hypertrophy, left ventricular diastolic dysfunction, and elevated left ventricular filling pressures despite preserved ejection fraction.

Cholangiocytes line the intrahepatic bile ducts and regulate salt and water secretion during bile formation, but the mechanism(s) regulating ductal water movement remains obscure. A water-selective channel, the aquaporin CHIP, was recently described in several epithelia, so we tested the hypothesis that osmotic water movement by cholangiocytes is mediated by CHIP. Isolated rodent cholangiocytes showed a rapid increase in volume in the presence of hypotonic extracellular buffers; the ratio of osmotic to diffusional permeability coefficients was > 10. The osmotically induced increase in cholangiocyte volume was inversely proportional to buffer osmolality, independent of temperature, and reversibly blocked by HgCl2. Also, the luminal area of isolated, enclosed bile duct units increased after exposure to hypotonic buffer and was reversibly inhibited by HgCl2. RNase protection assays, anti-CHIP immunoblots, and immunocytochemistry confirmed that CHIP transcript and protein were present in isolated cholangiocytes but not in hepatocytes. These results demonstrate that (i) isolated cholangiocytes and intact, polarized bile duct units manifest rapid, mercury-sensitive increases in cell size and luminal area, respectively, in response to osmotic gradients and (ii) isolated cholangiocytes express aquaporin CHIP at both the mRNA and the protein level. The data implicate aquaporin water channels in the transcellular movement of water across cholangiocytes lining intrahepatic bile ducts and provide a plausible molecular explanation for ductal water secretion.

Abstract Purpose: MV-CEA is an oncolytic measles virus currently being tested in patients with ovarian cancer and whose propagation can be monitored by measuring blood carcinoembryonic antigen (CEA) levels. MV-NIS is an oncolytic measles virus coding for the thyroidal sodium iodide symporter (NIS) whose propagation can be mapped by serial radioiodine imaging. Expression of both CEA and NIS genes from a single virus would combine sensitive, quantitative expression monitoring (CEA) with radioisotopic expression mapping (NIS). Because of the unfavorable replication kinetics of measles viruses expressing both CEA and NIS, we explored the feasibility of combining MV-CEA with MV-NIS for comprehensive virotherapy monitoring in ovarian cancer. Experimental Design and Results: Mice implanted with i.p. SKOV3ip.1 ovarian cancer xenografts received MV-CEA alone, MV-NIS alone, or a combination of MV-CEA plus MV-NIS. Viral gene expression was monitored by measuring blood CEA levels, and the location of virus-infected cells was monitored by gamma camera imaging. Surprisingly, mice receiving the combination of MV-CEA plus MV-NIS showed greatly superior responses to therapy, but this was associated with 10-fold lower plasma levels of CEA compared with mice treated with MV-CEA alone. In vitro studies showed superior replication kinetics of MV-NIS relative to MV-CEA. The gamma camera scans were considerably less sensitive than the plasma CEA marker for monitoring virus infection. Conclusions: Dual therapy with MV-CEA and MV-NIS is superior to treatment with either virus alone, and it allows noninvasive monitoring of virotherapy via soluble marker peptide and gamma camera imaging. This has important implications for the clinical development of oncolytic measles viruses.