IMMU-05. IMMUNOTHERAPY WITH RESIQUIMOD REPOLARIZES TUMOR-ASSOCIATED MYELOID CELLS AND IMPROVES EVENT-FREE SURVIVAL IN A TRANSGENIC MOUSE MODEL OF SONIC-HEDGEHOG (SHH) MEDULLOBLASTOMA

Abstract Resiquimod is a synthetic small molecule agonist of Toll-like receptors 7 and 8 (TLR-7/8) that modulates innate immune cells. We found TLR-7/8 are expressed in medulloblastoma exclusively by tumor-associated myeloid cells (TAMs). We tested whether systemically administered resiquimod modulated TAMs in a genetic Sonic hedgehog (SHH) medulloblastoma model, and whether this modulation would be therapeutically beneficial. We generated mice with medulloblastoma by crossing hGFAP-Cre and SmoM2 mouse lines. The resulting hGFAP-Cre/SmoM2 (G-Smo) mice developed medulloblastoma with 100% frequency and showed a median survival of 14.5 days (n=12). Treatment with 3 doses of resiquimod at postnatal days 10, 12 and 14 reduced tumor size and increased median survival to 37 days (n=10) (p=0.003508). Cellular studies showed that resiquimod altered TAM phenotype, rapidly inducing expression of the inflammatory marker VCAM1, and more slowly increasing TAM populations. Responses to the 3-dose regimen were ultimately limited by recurrence and all mice eventually died of tumor progression. Continued resiquimod therapy with every other day dosing was less effective than the 3-dose regimen, suggesting that TAM responses to resiquimod are dynamic and change with prolonged exposure. Our data show that innate immunity, mediated by TAMs and stimulated by TLR-7/8 agonist therapy, can produce a significant anti-tumor effect in medulloblastoma. The common expression of TLR-7/8 on TAMs in patient-derived medulloblastoma samples and in the mouse model suggests that resiquimod may produce similar anti-medulloblastoma effects in humans. Further studies are needed to define the mechanism of the anti-tumor effect in detail, to determine the optimal dose regimen, and to determine if resiquimod can combine effectively with additional adjuvant therapies to produce curative effects.

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IMMU-48. IMMUNOMODULATION WITH RESIQUIMOD REPOLARIZES THE IMMUNE MICROENVIRONMENT TO INHIBIT MEDULLOBLASTOMA PROGRESSION

Neuro-Oncology ◽

10.1093/neuonc/noab196.407 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi103-vi103

Author(s):

Christopher Park ◽

Coral Del Mar ◽

Morrent Thang ◽

Taylor Dismuke ◽

Duhyeong Hwang ◽

...

Keyword(s):

Tumor Growth ◽

Tumor Progression ◽

Median Survival ◽

Sonic Hedgehog ◽

Expression Patterns ◽

Myeloid Cells ◽

Pediatric Brain Tumor ◽

Toll Like Receptor ◽

Paracrine Signaling ◽

Pediatric Brain

Abstract BACKGROUND New, non-cytotoxic treatments may improve outcomes for medulloblastoma, the most common malignant pediatric brain tumor. Sonic hedgehog (SHH) subgroup medulloblastoma, which includes subtypes with poor prognosis, can be modeled in immunocompetent, transgenic mice. These models are ideal for preclinical testing of immunotherapies. Here, we show that immune stimulation using resiquimod, a small molecule agonist of Toll-like receptor 7 and 8 (TLR 7/8), alters myeloid populations in medulloblastoma and significantly reduces tumor growth. METHOD We generated mice with medulloblastoma by interbreeding the hGFAP-Cre and SmoM2 mouse lines. The resultant hGFAP-Cre/SmoM2 (G-Smo) mice develop SHH medulloblastoma with 100% frequency. We analyzed myeloid populations and demonstrated TLR7/8 expression patterns in G-Smo tumors. We then compared survival of untreated G-Smo mice versus G-Smo mice treated with three doses of resiquimod at postnatal days 10, 12, and 14. We also assessed pharmacodynamic effects at progressive intervals after a single dose. RESULTS Approximately 10% of cells in G-Smo medulloblastomas were myeloid cells, and these cells were the only cells that expressed TLR7/8. Resiquimod slowed tumor growth and increased the survival of mice with medulloblastoma. Untreated median survival was 14.5 days (n=12), compared to resiquimod-treated median survival of 37 days (n=10; p=0.0003). All treated mice eventually demonstrated tumor progression. Immunohistochemistry for IBA1, a pan-macrophage marker, demonstrated significant increase in myeloid cells within the tumor by 24 hours after treatment (p=0.0178), however the IGF1+ fraction of myeloid cells decreased (p=0.0275). CONCLUSION Resiquimod prolongs survival in mice with SHH-driven medulloblastoma, demonstrating the potential for therapies that target myeloid cells to produce significant anti-tumor effects. Myeloid-derived IGF-1 has been shown to support tumor progression and resiquimod may act by disrupting this paracrine signaling.

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Gfi136N As a Novel Marker and Therapeutic Target of MDS and AML

Blood ◽

10.1182/blood.v124.21.3245.3245 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3245-3245

Author(s):

Lars C. Michel ◽

Lacramioara Botezatu ◽

Judith Maria Hönes ◽

Anna Marneth ◽

Damien Grapton ◽

...

Keyword(s):

Mouse Model ◽

Hdac Inhibitors ◽

Leukemic Cells ◽

Healthy Population ◽

Epigenetic Changes ◽

Free Survival ◽

A Genome ◽

The Common

Abstract Growth Factor Independence 1 (GFI1) is a hematopoietic transcription factor that plays a crucial role in the development of myeloid precursor cells. Its variant single nucleotide polymorphism called GFI136N (whereby Serine at position 36 is replaced by Asparagine) has been described to play a role in acute myeloid leukemia (AML) development. The prevalence of this variant is about 5-7% in the healthy Caucasian populations. Patients with myelodysplastic syndrome (MDS) show disturbed bone marrow function and are at risk to develop AML. Identifying prognostic markers and potential targets is essential for improved MDS therapy. We explored how GFI136N influences onset and progression of MDS and investigated the biological mechanisms behind our findings. To examine the role of GFI136N with regard to progression of MDS to AML, we characterized the status of GFI1 in 201 German-, 350 US- and 86 Dutch MDS patients. Our results revealed that 11-13% of patients were heterozygous for GFI136N compared to 5-7% in the healthy population. Patients carrying GFI136N also showed a 2-3-fold higher risk of AML-development and inferior leukemia-free survival than patients carrying the common variant GFI136S. The onset of MDS and AML tended to be at younger ages for GFI136N carriers and they did not respond to demethylating therapy to the same extent as GFI136S homozygous MDS patients. Whole exome sequencing revealed that patients carrying GFI136N had mutations in epigenetic modifiers such as EZH2 and expressed higher levels of different oncogenes such as HOXA9. To dissect the mechanisms behind these findings and to study the role of GFI136N in AML development, we used GFI136N and GFI136S knock-in mice that expressed either of the two human variants instead of murine Gfi1. We transduced hematopoietic progenitor cells from GFI136S or GFI136N expressing mice with retroviral vectors overexpressing oncofusion proteins AML1-ETO9a or MLL-AF9, which can be recurrently found in AML patient cohorts. GFI136N expressing cells that had been transduced with either AML1-ETO9a or MLL-AF9 generated significantly more cells and colonies than GFI136S controls. In order to confirm our findings in vivo, we used three different AML and MDS mouse models crossed to mice carrying either GFI136N or the common GFI136S. As a first in vivoapproach, we used a mouse strain that expresses the human oncofusion protein CBFB-MYH11 (inv(16)) frequently found in AML patients. Mice carrying GFI136N and expressing CBFB-MYH11 showed a significantly accelerated onset of AML compared to mice carrying GFI136S (p=0.004). We found similar results in a second AML mouse model expressing MLL-AF9. Using the NUP98-HOXD13 MDS mouse model a marginally significant decrease in leukemia-free survival was observed in mice carrying GFI136N. To investigate molecular mechanisms that could cause the observed effects of GFI136N, we used ChipSeq and RNASeq. Our results revealed that GFI136N lineage negative cells show a genome-wide higher degree of H3K4 methylation and H3K9 acetylation than cells carrying GFI136S. GFI1 recruits among others LSD1 and HDAC to demethylate H3K4 and deacetylate H3K9 and thus GFI136N fails to initiate epigenetic changes previously shown for GFI1, leading to higher expression of various oncogenes such as HoxA9 and Kras. A number of MDS and AML patients are treated with HDAC and LSD1 inhibitors, but based on the above data we would predict that this therapy would likely fail in GFI136N patients and that therapy with histone acetylase (HAT) inhibitors or histone methyltransferase inhibitors would be more beneficial for these patients. To test this hypothesis in vitro, we treated the above-described GFI136N and GFI136S expressing AML1-ETO9a or MLL-AF9 leukemic cells with HAT or HDAC inhibitors. HAT inhibitors inhibited growth of preleukemic GFI136N expressing cells more efficiently than HDAC inhibitors. Similarly in vivo, we transplanted irradiated mice with GFI136N or GFI136S expressing MLL-AF9 leukemic cells. We found that HAT inhibitors tended to prolong the survival of mice transplanted with GFI136N leukemic cells more than mice transplanted with GFI136S leukemic cells. In summary, GFI136N is a novel prognostic marker for MDS patients that predisposes to AML likely via epigenetic changes at different oncogenes. We also suggest that HAT inhibitors could be more beneficial to GFI136N carrier patients thus enabling a more individualized therapy. Disclosures Platzbecker: TEVA: Research Funding, Speakers Bureau.

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24(S)-Saringosterol Prevents Cognitive Decline in a Mouse Model for Alzheimer’s Disease

Marine Drugs ◽

10.3390/md19040190 ◽

2021 ◽

Vol 19 (4) ◽

pp. 190

Author(s):

Nikita Martens ◽

Melissa Schepers ◽

Na Zhan ◽

Frank Leijten ◽

Gardi Voortman ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Mouse Model ◽

Cognitive Decline ◽

Inflammatory Marker ◽

Amyloid Β ◽

Liver Fat ◽

Gas Chromatography Mass Spectrometry ◽

Memory Decline ◽

Plaque Load

We recently found that dietary supplementation with the seaweed Sargassum fusiforme, containing the preferential LXRβ-agonist 24(S)-saringosterol, prevented memory decline and reduced amyloid-β (Aβ) deposition in an Alzheimer’s disease (AD) mouse model without inducing hepatic steatosis. Here, we examined the effects of 24(S)-saringosterol as a food additive on cognition and neuropathology in AD mice. Six-month-old male APPswePS1ΔE9 mice and wildtype C57BL/6J littermates received 24(S)-saringosterol (0.5 mg/25 g body weight/day) (APPswePS1ΔE9 n = 20; C57BL/6J n = 19) or vehicle (APPswePS1ΔE9 n = 17; C57BL/6J n = 19) for 10 weeks. Cognition was assessed using object recognition and object location tasks. Sterols were analyzed by gas chromatography/mass spectrometry, Aβ and inflammatory markers by immunohistochemistry, and gene expression by quantitative real-time PCR. Hepatic lipids were quantified after Oil-Red-O staining. Administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice without affecting the Aβ plaque load. Moreover, 24(S)-saringosterol prevented the increase in the inflammatory marker Iba1 in the cortex of APPswePS1ΔE9 mice (p < 0.001). Furthermore, 24(S)-saringosterol did not affect the expression of lipid metabolism-related LXR-response genes in the hippocampus nor the hepatic neutral lipid content. Thus, administration of 24(S)-saringosterol prevented cognitive decline in APPswePS1ΔE9 mice independent of effects on Aβ load and without adverse effects on liver fat content. The anti-inflammatory effects of 24(S)-saringosterol may contribute to the prevention of cognitive decline.

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Pathological Alternations of Mediastinal Fat-Associated Lymphoid Cluster and Lung in a Streptozotocin-Induced Diabetic Mouse Model

Microscopy and Microanalysis ◽

10.1017/s1431927620024824 ◽

2020 ◽

pp. 1-14

Author(s):

Yaser H.A. Elewa ◽

Osamu Ichii ◽

Teppei Nakamura ◽

Yasuhiro Kon

Keyword(s):

Endothelial Cells ◽

Lung Injury ◽

Mouse Model ◽

Immune Cells ◽

Inflammatory Markers ◽

Inflammatory Marker ◽

Diabetic Mouse ◽

Control Group ◽

Injury Progression

Diabetes is a devastating global health problem and is considered a predisposing factor for lung injury progression. Furthermore, previous reports of the authors revealed the role of mediastinal fat-associated lymphoid clusters (MFALCs) in advancing respiratory diseases. However, no reports concerning the role of MFALCs on the development of lung injury in diabetes have been published. Therefore, this study aimed to examine the correlations between diabetes and the development of MFALCs and the progression of lung injury in a streptozotocin-induced diabetic mouse model. Furthermore, immunohistochemical analysis for immune cells (CD3+ T-lymphocytes, B220+ B-lymphocytes, Iba1+ macrophages, and Gr1+ granulocytes), vessels markers (CD31+ endothelial cells and LYVE-1+ lymphatic vessels “LVs”), and inflammatory markers (TNF-α and IL-5) was performed. In comparison to the control group, the diabetic group showed lung injury development with a significant increase in MFALC size, immune cells, LVs, and inflammatory marker, and a considerable decrease of CD31+ endothelial cells in both lung and MFALCs was observed. Furthermore, the blood glucose level showed significant positive correlations with MFALCs size, lung injury, immune cells, inflammatory markers, and LYVE-1+ LVs in lungs and MFALCs. Thus, we suggest that the development of MFALCs and LVs could contribute to lung injury progression in diabetic conditions.

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Establishing Adrenocortical Carcinoma Specific Prognostic Genes Signature

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqaa161.324 ◽

2020 ◽

Vol 154 (Supplement_1) ◽

pp. S148-S149

Author(s):

A R Patil ◽

D S Dabrowski ◽

J Cotelingam ◽

D Veillon ◽

M Ong ◽

...

Keyword(s):

Median Survival ◽

Poor Prognosis ◽

Cancer Genomics ◽

Disease Free Survival ◽

Genetic Alterations ◽

Malignant Neoplasms ◽

Free Survival ◽

Signature Genes ◽

Disease Free ◽

Disease Specific

Abstract Introduction/Objective Adrenal Cortical Carcinoma (ACC) is a rare malignant neoplasms originating from adrenal cortical tissue with an annual incidence rate of 1 to 2 cases per million individuals. These tumors have poor prognosis with 5-year disease free survival being 30% after complete resection in Stage I to Stage III patients. Hence, there is a need for identifying prognostic markers for effective management of disease in these patients. Methods We analyzed the data in The Cancer Genome Atlas of 1141 ACC individuals, using cbioportal.org, a web- based platform for analysis of large-scale cancer genomics data sets, and derived correlation between prognosis and genetic alterations in approximately 51,309 genes. Results We identified 15 signature genes (NOTCH1, TP53, ZNRF3, LRP1, KIF5A, MDM2, LETMD1, MTOR, NOTCH3, RERE, SMARCC2, LDLR, HRNR, AVPR1A and PCDH15), alterations in which indicated a poor prognosis for ACC individuals. Analysis of 15 signature genes demonstrated that disease specific median survival for the patients with ACC, was reduced to 39.5 months (p value < 8 x 10 -9 and sensitivity of 93%) when any one or more of these genes was altered. Whereas, disease specific median survival was greater than 180 months (90% survival being 180 months) with no alteration in our signature genes. In addition, our analysis of our signature genes demonstrates reduced overall survival, disease free survival and progression free survival in individuals having alterations in our signature genes. Moreover, our set of 15 genes belonged mainly to MDM2-TP53, NOTCH and mTOR pathways, and small molecule modulators of these pathways are in process of development. Conclusion Our 15 gene signature was not only able to predict poor prognosis in ACC, but also has the potential to serve as a molecular marker set for initiation of NOTCH and mTOR specific targeted therapies in these patients.

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Crosstalk between Notch and Sonic hedgehog signaling in a mouse model of amyotrophic lateral sclerosis

Neuroreport ◽

10.1097/wnr.0000000000000725 ◽

2017 ◽

Vol 28 (3) ◽

pp. 141-148 ◽

Cited By ~ 15

Author(s):

Xiaoxing Ma ◽

Anna Drannik ◽

Fan Jiang ◽

Randy Peterson ◽

John Turnbull

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Mouse Model ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Sonic Hedgehog Signaling ◽

Lateral Sclerosis

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Investigating the Relationship between CD34+and CD3+ Cell Doses, One-Year Graft-Versus-Relapse-Free-Survival, Graft-Versus-Host Disease, and Overall Survival in Haploidentical Hematopoietic Stem Cell Transplantation: A Single Center Experience

Blood ◽

10.1182/blood-2019-129647 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2051-2051 ◽

Cited By ~ 1

Author(s):

Mindy Hsiao ◽

Anastasia Martynova ◽

George Yaghmour ◽

Chris Foss

Keyword(s):

Overall Survival ◽

Stem Cell ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Optimal Dose ◽

Relapse Free Survival ◽

Free Survival ◽

Improved Outcomes ◽

The Mean ◽

Related Mortality

Background: Haploidentical hematopoietic cell transplantation (haplo-HCT) has emerged as a popular alternative to traditional HLA-matched hematopoietic cell transplant. As the number of haplo-HCT's rises, investigating the factors that may affect outcomes is necessary in order to improve overall survival and reduce transplant-related mortality. The optimal dose of CD34+ cells used during haplo-HCT to ensure favorable outcomes using PTCy has not yet been reported though a range of 2 to 5.00x106 cells/kg is commonly used.Furthermore, the optimal dose of CD3+ cells is unknown however recent data has suggested less than 3.00x108 cells/kg may prevent the development of acute GVHD. The importance of studying the impact of CD34+/CD3+ cell dosing may help to improve outcomes in this setting. Methods: We retrospectively analyzed adult patients at USC Norris Cancer Hospital (age ≥ 21) who received haplo-HCT from 2014 to 2019. The primary end-point assessed was 1-year GVHD-free/relapse-free survival (GRFS) defined as grade 3-4 acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death in the first post-HCT year. Secondary end-points included 1-, 2-, and 3-year relapse-related mortality (RRM) and overall survival (OS) in addition to 1-year transplant related mortality (TRM) and incidence of both acute and chronic GVHD. Results: A total of 67 adult haplo-HCT recipients were reviewed. Of the patients evaluated, approximately 50% (n = 33) were male and 49% (n = 32) were female. The age range was 21-71 years old (median = 44), and the most common underlying hematologic disorders included AML (40%), ALL (38%), aplastic anemia (7.7%), and others (MDS, lymphoma, myelofibrosis, and HLH) (13.8%). 67% of patients received myeloablative conditioning regimens while 33% received reduced intensity regimens. 70% (n = 47) of patients received peripheral blood as a stem cell source with 30% (n = 20) receiving bone marrow. The mean CD34+ dose infused was 6.07x106 cells/kg and the mean CD3+ dose was 2.94x108 cells/kg. The mean time to recovery of platelets, neutrophils, and lymphocytes was 25, 18, and 37 days respectively. CD34+ stem cells ≥5.00x106 cells/kg was significantly associated with shorter time to lymphocyte recovery (p = 0.0265) though recovery less than 30 days was not significantly associated with OS (p = 0.5268). Incidence of 1-year GRFS was 71% (n= 46) and 1-, 2-, and 3-year RRM were 4.6%, 6%, and 7.7% respectively. 1-year TRM was 15.3% with 50% of deaths from acute GVHD. 1-, 2-, and 3-year OS were 80%, 78%, and 77% respectively. Factors significantly associated with increased mortality included use of RIC regimen (p = 0.004) and disease status at time of transplant (p = 0.04). Cumulative incidence of GVHD was 63% (n = 42) with 33% (n = 22) and 30% of patients (n = 20) with acute and chronic GVHD respectively. Lack of mild chronic GVHD was associated with increased mortality (p = 0.0029) and use of a myeloablative regimen (p = 0.0029) was significantly associated with GVHD. Subgroup analysis of those who received CD34+ dose ≥7.00x106 cells/kg (n = 24) and ≥10x106 cells/kg (n = 7) were found to have 1-year OS of 87.5% and 85.7% compared with 77% and 80% in those that received lower doses (p= 0.2229 and p = 1.00) respectively however this was not found to be significantly associated with increased incidence of GVHD, relapse, or mortality. Discussion: Our results demonstrate improved outcomes specifically 71% survived 1 year without experiencing at least 1 GRFS event compared with 24-35% reported by CIBMTR, Holtan et al 2015, and Solh et al 2016 with 3-year OS of 77% when compared with a previously reported 48%. The mean CD34+ cell dose of our population is higher than the standard range which may account for the improved outcomes however the dosing of CD34+/CD3+ cells were not significantly associated with our primary and secondary end-points. It was significantly associated, however, with shorter time to lymphocyte recovery, a factor that has been reported to be associated with decreased RRM and therefore improved OS. Furthermore, subgroup analysis of higher CD34+ dose did show a better 1-year OS though this was not statistically significant. Limitations of this study include small sample size and short follow-up period. Further research with a prospective study identifying the optimal CD34+/CD3+ cell dose in addition to comprehensive evaluation of immune recovery is warranted in order to improve haplo-HCT outcomes. Figure Disclosures Yaghmour: Jazz Pharmaceutical company: Consultancy, Speakers Bureau; Astella company: Speakers Bureau; Takeda: Speakers Bureau.

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Long-term disease-free survival in acute nonlymphocytic leukemia

Blood ◽

10.1182/blood.v57.6.1144.bloodjournal5761144 ◽

1981 ◽

Vol 57 (6) ◽

pp. 1144-1147

Author(s):

BA Peterson ◽

CD Bloomfield

Keyword(s):

Complete Remission ◽

Induction Chemotherapy ◽

Median Survival ◽

Cytosine Arabinoside ◽

Disease Free Survival ◽

Acute Nonlymphocytic Leukemia ◽

Maintenance Chemotherapy ◽

Free Survival ◽

Disease Free

Twenty-six of 45 adults (58%) with acute nonlymphocytic leukemia who were treated with intensive induction chemotherapy over 5 yr ago entered complete remission. All patients entering remission were placed on weekly maintenance chemotherapy consisting of cytosine arabinoside and 6-thioguanine. The median duration of complete remission was 17 mo and 7 patients (27%) remained in their initial remission for 62 + to 102 + mo. All but one of the patients in complete remission over 5 yr have had treatment discontinued. Only 1 of 7 patients in remission for more than 5 yr has relapsed. Median survival is 26.5 mo, and 8 patients (31%) currently remain alive without evidence of leukemia 63--105 mo from diagnosis. It is possible to achieve long-term disease-free survival with chemotherapy alone in acute nonlymphocytic leukemia.

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