Search

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Supplementary Figure S1 shows the cellular effects of AKIs and anti-leukemic agents across a panel of human AML cell lines. Supplementary Figure S2 shows expression of aurora-A and aurora-B protein levels in four AML cell lines. Supplementary Figure S3 shows AMG 900 induces a dose-dependent increase in polyploidy, apoptosis, and p53 protein levels in MOLM-13 cells. Supplementary Figure S4 shows AMG 900 plus Ara-C combination matrix and CI determination in MOLM-13 cells. Supplementary Figure S5 shows AMG 900 induced apoptosis is attenuated by peptide inhibitors of caspases in MOLM-13 cells. Supplementary Figure S6 shows FC gating strategy for annexin-V coupled JC-1 assay. Supplementary Figure S7 shows the anti-proliferative effects of AMG 900 and AZD1152-hQPA on primary human bone marrow mononuclear cells in culture. Supplementary Figure S8 shows a moderate reduction in mouse body weight after AMG 900 treatment.

BACKGROUND: Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma. METHODS: For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8(+) T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing. FINDINGS: Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks. INTERPRETATION: This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma. FUNDING: Regeneron Pharmaceuticals.

Supplementary Table 1 from Systemic Blockade of Transforming Growth Factor-β Signaling Augments the Efficacy of Immunogene Therapy

Supplementary Figure 1 from Systemic Blockade of Transforming Growth Factor-β Signaling Augments the Efficacy of Immunogene Therapy

Locally produced transforming growth factor-β (TGF-β) promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This article explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor SM16. Adenovirus expressing IFN-β (Ad.IFN-β) was injected intratumorally once in established s.c. AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a Kras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing human papillomavirus-E7 (HPV-E7; Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine expression and cell populations were measured in tumors and spleens by real-time PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.IFN-β increased the number of intratumoral leukocytes (including macrophages, natural killer cells, and CD8+ cells) and increased the percentage of T cells expressing the activation marker CD25. SM16 also augmented the antitumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the antitumor effects of Ad.IFN-β immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy. [Cancer Res 2008;68(24):10247–56

Background Immune checkpoint inhibitors (ICIs) can induce durable responses in multiple different tumor types. Lymphocyte activation gene-3 (LAG-3) is one of the immune checkpoints for which therapeutic antibodies are being developed. PET imaging with radiolabeled ICIs can visualize whether the drug reaches the tumor, and might serve as a target expression readout. Therefore, we aimed to evaluate the safety of the radiolabeled LAG-3 antibody 89Zr-DFO-REGN3767, to determine the optimal tracer dose and imaging time point, and to get insight into 89Zr-DFO-REGN3767 whole body distribution. Methods Patients with advanced solid tumors received 37 MBq 89Zr-DFO-REGN3767 together with unlabeled antibody to achieve a 2, 5, 10, 20, or 40 mg total protein dose (NCT04706715). Patients underwent PET/CT scans on days 0, 2, 4, and 7 after injection, followed by a tumor biopsy. After that, they received treatment with cemiplimab alone or combined with platinum-containing chemotherapy. PET scans were analyzed by placing volumes of interest (VOIs) in tumor lesions and normal tissues. Tracer uptake was measured in the VOIs and expressed as the maximum standardized uptake value (SUVmax) for tumor lesions and as the mean standardized uptake value (SUVmean) for normal tissues. Results The 16 included patients experienced no 89Zr-DFO-REGN3767-related side effects. Tumor-to-blood ratios for all dose levels increased from days 0 to 7. Therefore, imaging 7 days after tracer injection was deemed optimal to achieve the highest contrast. The 40 mg tracer dose resulted in the most favorable blood kinetics. At this dose, tracer uptake in tumor lesions (n = 17) varied between patients (geometric mean SUVmax 6.2; SD 1.7). In normal tissues, uptake was seen in the spleen (x̄ 11.2; SD 1.6), and bone marrow (x̄ 2.2; SD 0.7), which increased from days 0 to 7. Tracer uptake was visually present in regions with inflammation. Conclusion 89Zr-DFO-REGN3767 PET imaging is feasible and studied patients did not experience tracer related side effects. Optimal imaging results were achieved with the 40 mg protein dose and imaging on day 7 89Zr-DFO-REGN3767 shows specific accumulation in LAG-3 rich tissues and tumor lesions. Citation Format: Pim P. van de Donk, Lotte M. Smit, Joyce van Sluis, Wim Timens, Adrienne H. Brouwers, Marjolijn N. Lub- de Hooge, Sjoerd G. Elias, Jourik A. Gietema, Claudia A. van Winkel, JuAn Wang, Jason Giurleo, Thomas Uldrick, Hung Kam Cheung, Hilde Jalving, Sjoukje F. Oosting, Daan G. Knapen, Derk-Jan A. de Groot, Elisabeth G. de Vries. LAG-3 PET imaging in patients with cancer before immune checkpoint inhibitor therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5611.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B–selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein–expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3′-deoxy-3′-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Introduction: Hepatic resection is the accepted treatment of early-stage hepatocellular carcinoma (HCC), however, the majority of tumors recur despite surgery and no perioperative intervention has demonstrated a survival advantage. Neoadjuvant immunotherapy has shown success in inducing pathological responses alongside induction of immune memory in multiple tumor types and is being evaluated to determine its potential in HCC. Methods: We recently completed accrual into a trial of perioperative cemiplimab (anti-PD-1) for resectable HCC (NCT03916627, Cohort B). This trial enrolled 21 patients to receive 2 cycles of neoadjuvant cemiplimab (350 mg Q3W) followed by surgical resection and an additional 8 adjuvant cycles. The primary endpoint was significant tumor necrosis, defined as >70% necrosis of the resected tumor. Secondary endpoints included delay of surgery, overall response rate, adverse events (AEs), and change in lymphocyte infiltration. Patients underwent pre-treatment biopsies and regular blood collection throughout treatment to enable exploratory analyses including multiplex IHC and single-cell proteomic and transcriptomic analysis. The final patient enrolled underwent resection on December 28, 2020. Results: Over the course of 18 months, 21 patients were enrolled. All patients received 2 cycles of preoperative cemiplimab, and all but 1 patient underwent successful resection; 1 patient was found to have metastatic disease at the time of surgery and resection was aborted. Cemiplimab demonstrated an acceptable risk-benefit profile: 90.5% of patients experienced any treatment-emergent AE (TEAE) of any grade during the neoadjuvant treatment period through the perioperative period; 28.6% of patients experienced any Grade 3 TEAE. One patient experienced Grade 3 pneumonitis during neoadjuvant therapy and surgery was delayed by 2 weeks past protocol defined surgical window. Of the 20 patients with resected tumors, 4 (20%) met the predefined endpoint of >70% tumor necrosis and 7 (35%) had ≥50% tumor necrosis. Three (15%) of the 4 patients with >70% tumor necrosis had a pathological complete response, defined as 100% tumor necrosis. We observed similar patterns of change in degree of necrosis on pathological assessment and 3D MRI at baseline and following completion of immunotherapy. Initial pathological assessment suggests an association between immune cell infiltration and tumor response. Tissue analysis is ongoing and multiplex IHC relating myeloid and lymphoid infiltration patterns with response to cemiplimab will be presented at the meeting. Conclusion: This is the largest trial reported to date of neoadjuvant PD-1 targeted monotherapy in HCC. Pathological response data support larger trials to identify optimal clinical endpoints that correlate with improvement in survival, and to establish the utility and safety of perioperative PD-1 blockade. Citation Format: Thomas Urban Marron, Ganesh Gunasekaran, Parissa Tabrizian, Edward Kim, Maria Isabel Fiel, Stephen Ward, Zhen Zhao, Deborah Doroshow, Meredith Legg, Ashley Hammad, Paul Kennedy, Guray Akturk, Pauline Hamon, Antoinette Bonaccorso, Daniel Labow, Sacha Gnjatic, Bachir Taouli, Elizabeth Miller, Pradeep Thanigaimani, Nathalie Fiaschi, Jennifer Sims, Jayakumar Mani, Nicola James, Sara Hamon, Vladimir Jankovic, Siyu Li, Hung Kam Cheung, Gavin Thurston, Israel Lowy, Myron Schwartz, Miriam Merad. Neoadjuvant cemiplimab demonstrates complete pathological responses in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT182.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

We have constructed six new P-element-based Drosophila melanogaster transformation vectors that specifically allow for the high-level accumulation of any RNA of interest in the developing egg and pre-blastoderm embryo. Such specificity results, in part, from the inclusion in the vectors of an enhancer active exclusively in nurse cells, the principal providers of RNA to the egg and early embryo. The nurse cell enhancer was derived from the hsp26 heat-shock (HS) gene, but its activity was neither dependent on nor sensitive to HS. In addition to the nurse cell enhancer, two of the vectors contain sequences from the K10 gene that promote the early transfer of RNAs from nurse cells into the oocyte; RNAs that contain the K10 sequence are transferred into the oocyte during the early to middle stages of oogenesis (i.e., during stages 2-9), while RNAs that lack such sequences are stored in nurse cells until stage 11. All of the vectors contain a tsp and a multiple cloning site (MCS) immediately downstream from the hsp26 nurse cell enhancer. In three of the vectors, the MCS is preceded by an ATG start codon. A wild-type copy of the white gene is included in all of the vectors as a selectable marker for transformation. The specificity of the vectors was demonstrated by the analysis of the expression patterns of lacZ derivatives.

Locally produced transforming growth factor-β (TGF-β) promotes tumor-induced immunosuppression and contributes to resistance to immunotherapy. This article explores the potential for increased efficacy when combining immunotherapies with TGF-β suppression using the TGF-β type I receptor kinase inhibitor SM16. Adenovirus expressing IFN-β (Ad.IFN-β) was injected intratumorally once in established s.c. AB12 (mesothelioma) and LKR (lung cancer) tumors or intratracheally in a Kras orthotopic lung tumor model. Mice bearing TC1 (lung cancer) tumors were vaccinated with two injections of adenovirus expressing human papillomavirus-E7 (HPV-E7; Ad.E7). SM16 was administered orally in formulated chow. Tumor growth was assessed and cytokine expression and cell populations were measured in tumors and spleens by real-time PCR and flow cytometry. SM16 potentiated the efficacy of both immunotherapies in each of the models and caused changes in the tumor microenvironment. The combination of SM16 and Ad.IFN-β increased the number of intratumoral leukocytes (including macrophages, natural killer cells, and CD8+ cells) and increased the percentage of T cells expressing the activation marker CD25. SM16 also augmented the antitumor effects of Ad.E7 in the TC1 flank tumor model. The combination did not increase HPV-E7 tetramer-positive CD8+ T cells in the spleens but did induce a marked increase in the tumors. Tumors from SM16-treated mice showed increased mRNA and protein for immunostimulatory cytokines and chemokines, as well as endothelial adhesion molecules, suggesting a mechanism for the increased intratumoral leukocyte trafficking. Blockade of the TGF-β signaling pathway augments the antitumor effects of Ad.IFN-β immune-activating or Ad.E7 vaccination therapy. The addition of TGF-β blocking agents in clinical trials of immunotherapies may increase efficacy. [Cancer Res 2008;68(24):10247–56

The specification of cell fates along the dorsoventral axis of the Drosophila embryo is dependent on the asymmetric distribution of proteins within the egg and within the egg’s outer membranes. Such asymmetries arise during oogenesis and are dependent on multiple cell-cell interactions between the developing oocyte and its neighboring somatic follicle cells. The earliest known such interaction involves the generation of a signal in the oocyte and its reception in the follicle cells lying on the dorsal surface of the oocyte at ∼ stage 10 of oogenesis. Several independent lines of investigation indicate that the fs(1)K10 (K10) gene negatively regulates the synthesis of the signal in the oocyte nucleus. Here we present data that indicate that the accumulation of K10 protein in the oocyte nucleus is a multistep process involving: (1) the synthesis of K10 RNA in nurse cells, (2) the rapid transport of K10 RNA from nurse cells into the oocyte, (3) the localization of K10 RNA to the anterior margin of the oocyte, and (4) K10 protein synthesis and localization. K10 RNA is transported into the oocyte continuously beginning at ∼ stage 2. This indicates a high degree of selectivity in transport, since most RNAs synthesized in stage 2 and older nurse cells are stored there until stage 11, when nurse cells donate their entire cytoplasm to the oocyte. The sequences responsible for the early (pre-stage 11) and selective transport of K10 RNA into the oocyte map to the 3′ transcribed non-translated region of the gene. None of the other identified genes involved in dorsoventral axis formation are required for K10 RNA transport. Two such other genes, cappuccino (capu) and spire (spir) are, however, required for the subsequent localization of K10 RNA to the oocyte’s anterior end.

The highly organized structure of the Drosophila ovary makes it an ideal system for studying mechanisms of differential gene expression. Here we report the identification of a 171 bp sequence from the 5′ end of the hsp26 gene that functions as a female germ-line-specific transcriptional regulator when linked in two copies to a basal promoter. The regulator is active only in nondividing cells of the germ line, i.e., only in nurse cells and oocytes. It is not active in any examined tissue or cell type outside of the female germ line. Copper nuclease footprinting studies show that the germ line regulator contains two binding sites for each of two different ovarian nuclear factors. Point mutations in the DNA target sites of either nuclear factor abolish in vitro binding and in vivo transcriptional activity, indicating that each factor is a positive activator of nurse cell/oocyte transcription. The two factors may represent different classes of activator proteins, since an increase in the copy number of one factor’s DNA target site cannot compensate for a decrease in the copy number of the other factor’s target site.