SCID Beige Research Articles

Antitumor Activity of Dehydroxymethylepoxyquinomicin (DHMEQ) in Monotherapy and Combination with Cisplatin in the SKOV-3 Ovarian Cancer Model

The objective of this study was to evaluate the antitumor activity of DHMEQ as monotherapy and in combination with cisplatin in a human ovarian cancer xenograft model. Cisplatin was used as a comparator. To create the xenograft model, human ovarian cancer cells (SKOV-3 line) were subcutaneously implanted into immunodeficient mice. The study was conducted on female SCID Beige C.B-17 Cg-Prkdcscid Lystbg/Crl mice. Antitumor activity was determined by comparing tumor growth inhibition (TGI) in the treatment groups to that in the control group. Results showed that daily intraperitoneal administration of DHMEQ at a dose of 14 mg/kg following a single intraperitoneal dose of cisplatin at 4 mg/kg reduced tumor growth in the SKOV-3 cell line xenograft model.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Accumulating evidence indicates that ferroptosis, an iron-dependent cell death, is involved in the development of tumors, and targeting ferroptosis offers great potential in tumor therapy. However, the regulatory networks of ferroptosis in peripheral T cell lymphoma (PTCL) remain unclear. Methods: Paraffin-embedded tissues of 112 PTCL patients and 38 reactive hyperplasia cases were collected with informed consents. Lentiviral vectors were stably transfected into PTCL cell lines. Tandem mass tag (TMT)-mass spectrometry and RNA-sequencing (RNA-seq) were performed for downstream analysis. Co-IP and confocal immunofluorescence were performed to verify protein–protein interaction. Xenograft models were established using SCID Beige mice. Results: Firstly, both mRNA and protein levels of PTGDS were found to be higher in PTCL cells and patients’ tissue (Figure 1A–C). Survival analysis revealed that the positive expression of PTGDS was closely associated with worse prognosis in PTCL patients (Figure 1D). PTGDS overexpression could significantly promote the proliferation of PTCL cells while PTGDS knockdown inhibited it in vitro and in vivo (Figure 2A,B). Besides, PTCL cells with PTGDS knockdown displayed a higher apoptosis rate (Figure 2C). PTGDS specific inhibitor AT56 could significantly inhibit the proliferation of PTCL cells, in a dose- and time-dependent manner (Figure 2D). Moreover, AT56 treatment induced the cell cycle arrest at G0/G1 phase in PTCL cells (Figure 2E). Analysis based on TMT-mass spectrometry found that AT56 treatment significantly regulated the expression of ferroptosis-related molecules (Figure 3A) in PTCL cells. The anti-tumor effects of sorafenib and erastin, classic ferroptosis inducers, was significantly enhanced by AT56 treatment, and Fer-1, ferroptosis inhibitor, rescued the inhibitory role of AT56 on cell proliferation (Figure 3B,C). Moreover, AT56 promoted erastin/sorafenib-induced accumulation of lipid ROS (Figure 3D), a classic biomarker of ferroptosis. Further in-vivo experiments observed that AT56 treatment enhanced the anti-tumor effects of sorafenib (Figure 3E). Mechanically, structure analysis of differentially expressed molecules in TMT-mass spectrometry identified HMOX1, a regulator of heme catabolism, as PTGDS interactive protein. Co-IP and confocal immunofluorescence assays verified their co-localization and interaction in PTCL cells (Figure 4A,B). Moreover, AT56 treatment increased the level of intracellular iron, and HMOX1 knockdown could rescue it (Figure 4C). Keywords: aggressive T-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, molecular targeted therapies The research was funded by: National Natural Science Foundation (No. 82270200, No. 82170189, No. 82070203, No. 81800194, No. 81770210); Key Research and Development Program of Shandong Province (No. 2018CXGC1213); China Postdoctoral Science Foundation (No. 2021T1404223); Translational Research Grant of NCRCH (No. 2021WWB02, No. 2020ZKMB01); Shandong Provincial Natural Science Foundation (ZR2021YQ51); Taishan Scholars Program of Shandong Province; Shandong Provincial Engineering Research Center of Lymphoma; Academic Promotion Programme of Shandong First Medical University (No. 2019QL018). No conflicts of interests pertinent to the abstract.

NAT10 Promotes Tumorigenesis of Diffuse Large B-Cell Lymphoma By ac4C-Dependent Acetylation of SLC30A9

Introduction: N4-acetylcytidine (ac4C) is a chemical and conserved modification, which plays an essential role in the stability and regulation of mRNA translation. However, the role of RNA ac4C modification in diffuse large B-cell lymphoma (DLBCL) remains unclear. N-acetyltransferase 10 (NAT10), the key enzyme of ac4C modification, participates in various cellular processes. Herein, we explored the functional significance and regulatory mechanism of NAT10-mediated mRNA ac4C modification in the tumorigenesis of DLBCL, which is expected to propose novel therapeutic strategy. Methods: Lymph node biopsies from 104 newly diagnosed DLBCL patients and 20 reactive lymphoid hyperplasia were collected with informed consents. The biological function of NAT10 was explored through NAT10 knockdown (NAT10 KD) and CRISPR/Cas9 mediated genomic deletion (NAT10 KO). RNA sequencing and acRIP-seq were performed to detect the dysregulated RNA in DLBCL. LC-MS/MS, RIP assay and RNA stability assay were performed to investigate the functional mechanism of NAT10. SCID beige mice were subcutaneously injected into LY1 cells to establish DLBCL xenograft model. Animal experiments were performed in accordance with the principles of the Institutional Animal Care. Results: We first evaluated the expression level of NAT10 in DLBCL as well as analyzed the correlation between NAT10 expression and clinicopathologic characteristics. Upregulation of NAT10 mRNA were identified in DLBCL. Survival analysis revealed that high NAT10 expression was associated with poor prognosis of DLBCL patients. In addition, high expression levels of NAT10 protein were validated in DLBCL cell lines and newly diagnosed DLBCL patients. To verify the biological functions of NAT10, we deleted NAT10 expression by CRISPR/Cas9 mediated genomic-editing system. Deletion of NAT10 induced cell cycle attest in G0/G1 phase. Knockdown of NAT10 resulted in similar anti-lymphoma effects. We further investigated the activities of NAT10 inhibition in DLBCL xenograft mice model. The results revealed that deletion of NAT10 significantly suppressed proliferation of DLBCL cells. RNA sequencing and acRIP-seq of LY1 cells with NAT10 knockout were performed to explore the mechanism of NAT10 in DLBCL. Knockout of NAT10 reduced the enrichment of ac4C peaks and significantly down-regulated the expression of Solute Carrier Family 30 Member 9 (SLC30A9) mRNA levels through integrative analysis of the sequencing results (Figure 1). RIP-qPCR further verified the combination of SLC30A9 and NAT10. Notably, SLC30A9 was downregulated upon NAT10 knockout, and positively correlated with NAT10 expression, suggesting that SLC30A9 may act as a feasible downstream target of NAT10 in DLBCL. Given that NAT10 is an RNA acetyltransferase, we next explored whether NAT10-mediated expression of SLC30A9 was dependent on ac4C modification. Lower global ac4C level was showed in DLBCL cells with NAT10 knockout. Analysis of SLC30A9 mRNA sequence found that the vast majority of ac4C peaks were distributed in the protein-coding region. Moreover, the NAT10 mut plasmid without a functional RNA helicase domain (K290A) or acetyltransferase domain (G641E) was constructed to explore the regulatory mechanism of NAT10 on SLC30A9 expression. Overexpression of NAT10 wild-type (WT) but not NAT10 mutant induced a significant increase in SLC30A9 expression levels. Furthermore, overexpression of NAT10 WT significantly increased the half-life of SLC30A9 mRNA (Figure 2). The above results suggested that SLC30A9 was regulated by NAT10 in an ac4C-dependent manner. Conclusions: Taken together, this study provides preclinical evidence for the significance of NAT10 in lymphomagenesis and highlights the regulatory mechanism of NAT10-mediated the expression of SLC30A9 in an ac4C-dependent manner in DLBCL. Further investigations on the specific NAT10 inhibitors in DLBCL will outline a promising treatment approach to target this newly identified oncogene in DLBCL therapy. Keywords: Diffuse large B-cell lymphoma (DLBCL), N-acetyltransferase 10 (NAT10), Solute Carrier Family 30 Member 9 (SLC30A9), CRISPR/Cas9 Disclosure: No relevant conflicts of interest to declare. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Abstract IA023: Therapeutic efficacy of trametinib and ganitumab in RAS-mutated rhabdomyosarcoma

Abstract Background: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS75807, but this combination was not pursued clinically due to excessive toxicity in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor that would be better tolerated in murine models and effective in both cell line and patient derived xenograft models of RAS-mutant FN RMS. Methods: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a monoclonal antibody with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of the combination was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in five out of six models of RAS-mutant RMS. Evidence suggests that the combination had little effect on body weight loss, thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the AKT phosphorylation that is induced by MEK inhibition alone. Therapeutic response to the combination was observed in models with an intact PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. The trametinib/ganitumab combination also likely has an improved tolerability profile compared to other IGF1R/MEK inhibitor combinations. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS. Citation Format: Marielle E. Yohe, Katie E. Hebron, Xiaolin Wan, Jacob S. Roth, David J. Liewehr, Nancy E. Sealover, Stacey Stauffer, Olivia Feehan-Nelson, Wenyue Sun, Kristine A. Isanogle, Christina M. Robinson, Amy James, Parirokh Awasthi, Priya Shankarappa, Xiaoling Liu, Haiyan Lei, Donna Butcher, Roberta Smith, Elijah F. Edmonson, Jin-Qui Chen, Noemi Kedei, Cody S. Peer, Jack F. Shern, W. Douglas Figg, Lu Chen, Matthew D. Hall, Simone Difillipantonio, Frederic G. Barr, Robert L. Kortum, Angelina V. Vaseva, Javed Khan. Therapeutic efficacy of trametinib and ganitumab in RAS-mutated rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA023.

Design and Characterization of Immune-Stimulating Imidazo[4,5-c]quinoline Antibody-Drug Conjugates.

Traditional antibody-drug conjugate (ADC) technology has employed tumor-targeting antibodies to selectively deliver ultrapotent cytotoxins to tumor tissue. While this technology has been highly successful, resulting in the FDA approval of over 10 ADCs, the field continues to struggle with modest efficacy and significant off-target toxicity. Concurrent with the struggles of the ADC field, a new generation of immune-activating therapeutics has arisen, most clearly exemplified by the PD-1/PD-L1 inhibitors that are now part of standard-of-care treatment regimens for a variety of cancers. The success of these immuno-oncology therapeutic agents has prompted the investigation of a variety of new immuno-stimulant approaches, including toll-like receptor (TLR) activators. Herein, we describe the optimization of ADC technology for the selective delivery of a potent series of TLR7 agonists. A series of imidazole[4,5-c]quinoline agonists (as exemplified by compound 1) were shown to selectively agonize the human and mouse TLR7 receptor at low nanomolar concentrations, resulting in the release of IFNα from human peripheral blood mononuclear cells (hPBMCs) and the upregulation of CD86 on antigen-presenting cells. Compound 1 was attached to a deglycosylated (Fc-γ null) HER2-targeting antibody via a cleavable linker, resulting in an ADC (anti-HER2_vc-1) that potently and selectively activated the TLR7 pathway in tumor-associated macrophages via a "bystander" mechanism. We demonstrated that this ADC rapidly released the TLR7 agonist into the media when incubated with HER2+ cells. This release was not observed upon incubation with an isotype control ADC and furthermore was suppressed by co-administration of the naked antibody. In co-culture experiments with HER2+ HCC1954 cells, this ADC induced the activation of the NFκB pathway in mouse macrophages and the release of IFNα from hPBMCs, while a corresponding isotype control ADC did not. Finally, we demonstrated that IP administration of anti-HER2_vc-1 induced complete tumor regression in an HCC1954 xenograft study in SCID beige mice. Unlike related ADC technology that has been reported recently, our technology relies on the passive diffusion of the TLR7 agonist into tumor-associated macrophages rather than Fc-γ-mediated uptake. Based on these observations, we believe that this ADC technology holds significant potential for both oncology and infectious disease applications.

Aberrant SPOP-CHAF1A ubiquitination axis triggers tumor autophagy that endows a therapeutical vulnerability in diffuse large B cell lymphoma

PurposeAberrant epigenetic changes, like DNA methylation, histone modifications, or ubiquitination, could trigger metabolic disorders in human cancer cells. This study planed to uncover the biological roles of epigenetic SPOP/CHAF1A axis in modulating tumor autophagy during Diffuse large B-cell lymphoma (DLBCL) tumorigenesis.Materials and methodsThe Immunohistochemistry (IHC) was performed to assess the CHAF1A expressions. The expression data of CHAF1A was derived from The Cancer Genome Atlas (TCGA), GSE32918 and GSE83632 datasets. Bioinformatic assays contain differential analysis, functional enrichment analysis and Kaplan–Meier survival curve analysis. The colony generation assay, Transwell assay and CCK-8 assays were conducted for the in vitro assays. The in vivo ubiquitination assays were used to assess regulations of SPOP on CHAF1A. The Chromatin immunoprecipitation (ChIP) assays were used to uncover epigenetic regulations of CHAF1A on TFEB. The relevant DLBCL cells were subcutaneously injected to SCID beige mice to establish the xenograft models.ResultsBioinformatic results revealed that CHAF1A expressed highly in DLBCL that were validated in patients samples. Patients with high CHAF1A suffered from inferior prognosis with shorter survival months relative to those with low CHAF1A. High CHAF1A enhanced DLBCL aggressiveness, including cell proliferation, migration and in vivo growth. Mechanistically, E3 ubiquitin ligase SPOP binds to and induces the degradative ubiquitination of CHAF1A via recognizing a consensus SPOP-binding motif in CHAF1A. SPOP is down-regulated in DLBCL and habours two DLBCL-associated mutations. Deficient SPOP leads to accumulated CHAF1A proteins that promote malignant features of DLBCL. Subsequently, ChIP-qPCR assay revealed that CHAF1A directly binds to TFEB promoters to activate the expressions. High CHAF1A could enhance the transcriptional activity of TFEB and downstream genes. The SPOP/CHAF1A axis modulates TFEB-dependent transactivation to regulate the lysosomal biogenesis and autophagy. The in vivo models suggested that TFEB inhibition is effective to suppress growth of SPOP-deficient DLBCLs.ConclusionsCHAF1A is aberrantly elevated in SPOP-deficient DLBCL. The in‐depth mechanism understanding of SPOP/CHAF1A/TFEB axis endows novel targets for DLBCL treatment.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P > 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

Improving Fat Graft Retention with Micro/Nanobubbles in Vivo

PURPOSE: Fat grafting is one of the most popular procedures in plastic and reconstructive surgery.1 However, retention rates of transplanted fat remain variable, often rendering unpredictable outcomes in the long term. Inadequate tissue oxygenation during the transplant process is thought to play a major role in this variability.2 To that end, we sought to examine the implementation of oxygenated micro/nanobubbles (MNBs) during transplantation of lipoaspirate as a means of maintaining adequate tissue oxygenation and improving graft survival. MNBs are small gas bubbles (<100 µm) that are stable for hours and can be saturated with high amounts of oxygen.3 Furthermore, their negative charge and irregular surface characteristics make them an ideal agent for separation and decontamination of charged particulate matter.4 We hypothesize that MNBs will enhance lipoaspirate survival and establish themselves as an important adjunctive step to be incorporated into current fat grafting techniques. METHODS: Twelve 6-week-old Fox Chase SCID beige mice were used as hosts for transplanted human lipoaspirate. Lipoaspirate samples harvested from healthy human donors were washed with either an oxygenated MNB or saline solution prior to injection into the dorsum of the mice. To assess graft viability, explants were harvested at 4-, 8-, and 12-week intervals. Following harvest, grafts were weighed, and volumes were obtained using gas pycnometry. Immunohistochemistry was completed utilizing antibodies directed toward CD31, Perilipin, and CA-9 as surrogates for angiogenesis, adipogenesis, and hypoxia, respectively. Quantitative analysis of IHC images was performed via ImageJ. RESULTS: The grafts that were washed in the MNB solution were significantly greater by mass as early as 4 weeks (P < 0.01). Likewise, an analysis of variance (ANOVA) showed that MNB-washed explants had greater volumes across all time points (P < 0.05). While CD31 staining showed that vessel density was equivocal at each interval between experimental and control groups, perilipin staining showed significantly greater intensity in the MNB group at both 4 and 8 weeks. Moreover, CA-9 staining intensity in the MNB group was notably lower by 12 weeks compared with that in the control group. CONCLUSIONS: The utilization of MNBs, as a source of oxygen, in the wash step prior to transplant may be beneficial for improving graft survival. MNB-washed grafts displayed greater volumes and masses over 12 weeks compared with their control-group saline-washed counterpart. Furthermore, as evidenced by the surrogate markers in the IHC analysis, lipoaspirate samples subjected to the MNB-wash demonstrated improved de novo adipogenesis and less hypoxia. Taken together, these preliminary data reveal promising translatable implications for oxygenated MNBs in the future of fat grafting paradigms. REFERENCES: 1. Simonacci F, Bertozzi N, et al. Procedure, applications, and outcomes of autologous fat grafting. Ann Med Surg (Lond). 2017;20:49–60. 2. Khouri RK, Jr., Khouri RE, et al. Diffusion and perfusion: the keys to fat grafting. Plast Reconstr Surg Glob Open. 2014;2(9):e220. 3. Matsuki N, Ichiba S, et al. Blood oxygenation using microbubble suspensions. Eur Biophys J. 2012;41(6):571–578. 4. Sayadi LR, Ziegler ME, et al. Topical oxygen therapy & micro/nanobubbles: a new modality for tissue oxygen delivery. Int Wound J. 2018;15(3):363–374.

Germinal Center Kinase As a Therapeutic Target in Multiple Myeloma

Introduction: Despite many new drug approvals, Multiple Myeloma remains an incurable disease due to development of drug resistance. This process is driven by clonal evolution in which kinases play a major role. Germinal center kinase (GCK) is the founding member of the GCKs, a group of Ser/Thr kinases homologous to the saccharomyces cerevisiae Ste20p, and participates in B cell differentiation. Among human tissues, GCK is highly expressed in transformed lymphocytes, spleen and whole blood. However, the expression and function of GCK in human myeloma is totally unknown.Methods and Results: First we analyzed the expression level of GCK in human MM cells and found that GCK is abundantly expressed in all the MM cell lines we tested. To determine whether GCK is required for MM cell growth, we introduced inducible shRNA to knock down GCK expression in MM cells by lentiviral mediated transduction. MM cell lines MM.1S and RPMI-8266 were stably transduced with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced GCK-tet-on-shRNA expression resulted in GCK protein knockdown and significant inhibition of cell growth by 75% in WST-1 proliferation assay. In accordance, cell cycle analysis revealed that the knockdown of GCK decreased cells in S-phase with concomitant increase in G0/G1 growth arrest (55% vs 67%). Importantly, GCK knockdown resulted in significant increase (3.21% vs 60.8%) of apoptotic cells suggesting that inhibition of GCK is a potential therapeutic target in MM cells. This was further confirmed by treating MM cells lines (MM.1S, RPMI-8266 and H929) with GCK inhibitors TL4-12, NG25 and Bay 61-3606. We found that these drugs dose dependently and significantly inhibit multiple myeloma cell growth, with IC50 7, 1.6 and 0.5 µM, respectively in H929 cells by WST-1 proliferation assay. Mechanistic studies showed that Bay 61-3606 dose-dependently decreases the protein levels of IKZF1/IKZF3 and c-Myc, increases p53 level and induces PARP cleavage resulting in cell cycle G1 arrest (60.8 vs. 69.8 with 1 µM) and apoptosis (9.16 vs. 29.4 at 1 µM) in MM.1S cells. To confirm the critical role of GCK in in vivo growth of MM cells, we generated subcutaneous MM xenografts in SCID Beige (CB17.Cg-PrkdcscidLystbg-J/Crl) mice using doxycycline-inducible GCK shRNA MM.1S cells. Doxycycline or vehicle treatment was started after the tumor was established on day 16. In contrast to vehicle-treated MM.1S-tet-on-shGCK or doxycycline-treated MM.1S-tet-on-sh-control tumors, doxycycline treated animals bearing MM.1S-tet-on-shGCK xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 30 days. Immunohistochemistry staining of tumors confirmed the decrease of GCK and IKZF1 expression in doxycycline-treated mice bearing MM.1S-tet-on-shGCK tumors compared with tumors of vehicle- or non-doxycycline treated mice.Conclusion: Our findings show that GCK is a potential target for multiple myeloma therapy. GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation. Furthermore, the inhibition of in vivo tumor growth correlates with the doxycycline-induced GCK knockdown, further confirming the critical role of GCK in MM tumorigenesis. DisclosuresLentzsch:Amgen: Consultancy; BMS: Consultancy; Caelum Biosciences: Other: leadership position and stock.

Combination of IRAK4 Inhibitor CA-4948 with BCL2 Inhibitor Venetoclax Induces Tumor Regression in an ABC-DLBCL Xenograft Model

IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein, which is mutated in ~22% of diffuse large B-cell lymphoma (DLBCL) cases. The MYD88- L265P activating mutation is found in ~30% of the activated B-cell (ABC) and ~6% of germinal center B-cell (GCB) subtypes of DLBCL and leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. In Waldenstrom macroglobulinemia (WM), the MYD88- L265P activating mutation is present in >90% of cases. Thus, the development of small molecule inhibitors targeting IRAK4 is an attractive anticancer strategy for MYD88 mutation-containing cancers such as DLBCL and WM.We are developing a novel IRAK4 inhibitor, CA-4948, as a therapeutic agent for non-Hodgkin lymphoma (NHL) and hematological cancers with dysregulated TLR/MYD88/IRAK4 signaling. CA-4948 is a reversible kinase inhibitor that modulates IRAK4 function in both the toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling cascades, and demonstrates pharmacodynamic and antitumor activity in in vitro and in vivo nonclinical models. CA-4948 exhibits favorable DMPK properties, oral bioavailability, and is well tolerated in mice. Furthermore, CA-4948 was previously shown to exhibit dose-dependent efficacy in ABC-DLBCL MYD88 -L265P xenograft tumor models using cell lines OCI-LY3 and OCI-Ly10 and patient-derived tumors.Activating mutations in the B-cell receptor (BCR) signaling pathway are frequently present in various NHL subtypes, leading to activation of NF-kB signaling, and growth and survival pathways. Dysregulation of both TLR and BCR pathways suggest that targeting both pathways will be required for improved therapeutic responses in NHL. Here, we report efficacy results from combined treatment of CA-4948 and the BCL2 inhibitor venetoclax in an ABC-DLBCL xenograft tumor model.We performed an in vivo study in female SCID Beige mice implanted with the ABC-DLBCL cell line OCI-Ly10, which harbors TLR (MYD88 -L265P) and BCR (CD79A ITAM) pathway activating mutations. OCI-Ly10 tumor-bearing mice were treated with CA-4948 (50 mg/kg, qd, po), venetoclax (75 mg/kg, qd, po) or the combination for 21 continuous days. Single agent treatment of CA-4948 and venetoclax exhibited moderate tumor growth inhibition (TGI) of 63% and 71%, respectively, and the combination demonstrated tumor regression. The combination was tolerated at the end of treatment without body weight loss relative to predose weight. After the 21-day drug treatment period, the mice were given a 19-day dosing holiday. Within 5 days of the holiday rapid tumor growth occurred in the previously treated single drug-treated mice and this rapid growth continued for the remainder of the dosing holiday. In contrast, tumors from the combination drug-treated mice did not commence detectable regrowth until the 10th day of the drug holiday and proceeded with a slow growth rate for the subsequent 9 days.After the 19-day drug holiday, dosing of the respective drug treatments was reinstated to each cohort. After 7 days of re-dosing, the tumors in each cohort exhibited antitumor responses to respective drug treatments with the CA-4948 plus venetoclax combination again driving the tumors into regression. In summary, CA-4948 plus venetoclax was a well-tolerated drug combination that exhibited a much greater in vivo antitumor response as compared to the single agent treatments. These results underscore the therapeutic potential of targeted IRAK4 kinase inhibition by CA-4948 in combination with other targeted agents for the treatment of NHL. DisclosuresBooher:Curis, Inc: Employment, Equity Ownership. Dellarocca:Curis, Inc: Employment, Equity Ownership. Atoyan:Curis, Inc: Employment, Equity Ownership. Borek:Curis, Inc: Employment, Equity Ownership. Samson:Curis, Inc: Employment, Equity Ownership. Tuck:Curis, Inc: Employment, Equity Ownership.

Combined Targeting of the Proteasome and Bcl-2 Sensitizes Human B-Cell Lymphoma and Multiple Myeloma to Cyclophosphamide in In Vitro and In Vivo Models of These Diseases.

The balance of apoptotic proteins is a critical mechanism used by the cell to maintain the mitochondrial transmembrane potential, which plays a critical role in governing apoptosis. Bortezomib (B) is a proteasome inhibitor that selectively inhibits the 26S proteasome, leading to an increase in many intracellular proteins, including proteins that lead to inhibition of NF-kB, as well as proapoptotic proteins like bik and Bax. Oblimersen (Obl) is a 18 mer oligonucleotide results in decreased transcription of the antiapoptotic protein bcl-2. We hypothesized that the mitochondrial membrane potential could be influenced by the co-administration of these agents to favor aopotosis. We report results of pre-clinical in vitro and in vivo experiments using these agents in combination in various lymphoma (NHL) & myeloma (MM) models. NHL & MM cell lines were exposed to Obl in the presence of cationic lipid and incubated for 48 hours. DNA synthesis & cell growth arrest was quantified by 3H-thymidine incorporation assays at 72 hrs, & revealed a concentration dependent cell killing. Induction of apoptosis in the presence or absence of different concentrations of B was detected by multiparameter flow cytometric analysis. Data reveals addition of bortezomib, even at relatively low concentrations (10 nM), additively improved the effects of Obl in all the cell lines studied. A SCID beige model of aggressive large B-cell lymphoma was used by injecting 1x10e7 cells (SKI-DLCL-1 or RL, a cell line over expressing bcl-2 through the 14:18 translocation) as a subcutaneous flank tumor. In over 6 in vivo experiments, the combination of B (0.5 mg/kg days 3 & 7) + Obl (3 mg/kg days 1,3,5, &7) consistently resulted in about 50% inhibition of growth of both SKI-DLCL-1 & RL compared to that in the control. Surprisingly, results were evident even using a low dose schedule of bortezomib (about one quarter of the MTD), suggesting the possibility of a small sensitizing dose of this drug. In all experiments, growth delay seen with the doublet exceeded the results noted with any singlet (B or Obl alone). Integration of cyclophosphamide at a dose of 50 mg/kg produced marked tumor shrinkage that appeared synergistic in several circumstances. Single agent CTX produced excellent growth delay (similar to any doublet), but in all these cases, tumor re-growth was evident by 21–25 days post tumor transplant. CTX dose & schedule was also important. Same day ‘lump and dump' of all drugs failed to effect cured, while CTX preceding B consistently produced best results, with cures found only in this triplet cohort. Results suggest that optimal scheduling of agents requires a ‘lead in' exposure of Obl, followed by CTX preceding all doses of bortezomib. These studies support the efficacy of this regimen.

Targeting Antiapoptotic Bcl-2 Family Members with AT-101 in Pre-Clinical Models of Aggressive Lymphoma in Combination with Cyclophosphamide (C) and Rituximab (R) Produces a Marked Improvement in Therapeutic Efficacy.

Over-expression of anti-apoptotic members of the Bcl-2 family, including Bcl-2 and Bcl-XL, have been observed in more that 80% of B-cell Lymphomas. AT-101 has recently been found to be a potent small molecule inhibitor of Bcl-XL and Bcl-2. We investigated the toxicity and antitumor activity of oral AT-101 in SCID beige mice following subcutaneous injection with 1 x 107 RL-DLBCL cells. In the first experiment we explored four daily (25, 50, 75, 100 mg/kg) and three weekly (200, 240, 280 mg/kg) dose levels of AT-101 in cohorts of 5 mice. The 25 mg/kg/day for 4 weeks schedule exhibited the safest profile with some tumor growth delay compared to the control. Higher daily doses of 50, 75 and 100 mg/kg/day were associated with significant weight lost (i.e. > 10% of the initial weight) after 14, 10 and 7 days respectively, with death of all animals in these dose groups by day 23, 21 and 16 respectively. The weekly schedules exhibited later onset toxicity with the 240 mg/kg cohort exhibiting the best reduction in tumor growth compared to control (p=0.063). In a second single agent experiment additional schedules were explored to try to improve the toxicity profile. These schedules included 100, 120, 140 mg/kg on days 1, 4, 8, 11 followed by 1 week; a 35 and 50 mg/kg/day for 2 weeks out of 4; a 100 mg/kg for 3 days/week for 3 weeks out of 4; and a 240 and 280 mg/kg/week for 3 weeks out of 4. Significant weight loss was noted at 5–7 days in the dose groups at 100 mg/kg daily, 120–140 and 280 mg/kg. Overall, tumor volume assessment showed a statistically significant advantage for the 35 mg/kg daily schedule compared to control (p=0.008), with this dose and schedule being the most effective and least toxic. In terms of dose intensity, it's interesting to note that 35 mg/kg/day for two weeks delivered a total dose of 490 mg/kg. Combination experiments with cyclophosphamide (50 mg/kg) and rituximab (10 mg/mg) i.p. on days 2, 4, 6, 8 demonstrated marked improvement in favor of the triplet combination (see Fig. below). AT-101 (35 mg/kg) was administered p.o. from day 0 to day 9 These data suggest that targeting Bcl-2 and Bcl-XL with AT-101 may help chemosensitize lymphoma to conventional therapies and improve therapeutic outcomes. [Display omitted]

Cisplatin Loaded Poly(L-glutamic acid)-g-Methoxy Polyethylene Glycol Complex Nanoparticles Combined with Gemcitabine Presents Improved Safety and Lasting Anti-Tumor Efficacy in a Murine Xenograft Model of Human Aggressive B Cell Lymphoma.

Cisplatin (CDDP) is a highly effective anti-tumor drug with a broad spectrum of activity. However, the clinical efficacy of CDDP-containing regimens is yet unsatisfactory due to the severe dose-related toxicity of CDDP. In a previous study, CDDP nanoparticles (L-CDDP) forms a complex as CDDP with poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) with improved safety compared to CDDP. Herein, a murine xenograft model of human aggressive B cell lymphoma (BCL) was established to explore anti-lymphoma efficiency of L-CDDP combined with GEM. BJAB cells represent an aggressive BCL, which were utilized to explore the anti-proliferative effect, cell apoptosis via CCK-8 test and flow cytometry technology, respectively. Toxicity experiment and the maximum tolerated dose (MTD) test were conducted in Kunming mice. Tumor inhibition experiment was conducted at the dose of MTD in SCID beige mice-bearing lymphoma. In this study, the loading capacity and encapsulating efficiency of CDDP in the L-CDDP was 18.3% and 89.7%, respectively, and the hydrodynamic diameter of the prepared L-CDDP was 20.6 nm. The CCK-8 data indicated that the anti-proliferative activity of monodrug groups (GEM, CDDP, L-CDDP) was dose- and time-dependent in BJAB cells. The synergistic effects in anti-lymphoma were detected in these two groups (GEM+CDDP, GEM + L-CDDP). Compared to control group, the proportion of apoptotic cells in experimental groups in BJAB cells was significantly higher at 48 h. Toxicity assays revealed that GEM + L-CDDP regimen had low hematological toxicity, hepatotoxicity, and nephrotoxicity. Tumor inhibition experiment demonstrated that GEM + L-CDDP group exhibited significant tumor-suppressing effects. Moreover, tumors continued to shrink in GEM + L-CDDP group, while these appeared to grow in the GEM + CDDP group. Finally, tumor necrosis was most prominent in the GEM + CDDP and GEM + L-CDDP groups, as assessed by hematoxylin-eosin staining. In conclusion, compared to CDDP, L-CDDP combined with GEM seriously induces BJAB cell apoptosis. Also, GEM + L-CDDP exhibits low hematotoxicity, hepatotoxicity, and nephrotoxicity. Importantly, GEM + L-CDDP presents lasting anti-lymphoma efficacy in a SCID beige mice-bearing lymphoma.

Effects of Tripterine on Adhesion Molecules and Cell Cycle in Human Acute Promyelocytic Leukemia Model Mice

To observe the effects of tripterine on adhesion molecules and cell biological characteristics in mice with acute promyelocytic leukemia (APL) tumor. Eighteen SCID beige mice were caudal vein injected with NB4 cell lines (5×106/only) to construct a human APL tumor-bearing model, then the mice were divided into tumor-bearing model group, arsenic trioxide group and tripterine group randomly, and another 6 mice which didn't construct model were set up as control group; after 3 weeks, the control group and the tumor-bearing model were intraperitoneally injected with normal saline as compared, arsenic trioxide was intraperitoneally injected according to 100 μg/kg, and tripterine was intraperitoneally injected according to 3 mg/kg. Four groups were all injected for 3 weeks. The biological characteristics of NB4 cells and the expression of adhesion molecules in venous blood of mice after treatment were observed. The neutrophil decrased and promyelocytes, NB4 cells, B lymphocytes and white blood cells increased in tumor-bearing group as compared with control group (P<0.05), and the expressions of serum P-selectin (P-selectin), soluble vascular adhesion molecule-1 (soluble vascular adhesion molecule-1, sVCAM-1) and soluble intercellular adhesion molecule-1 (soluble intercellular adhesion molecule-1, sICAM-1) all increased (P<0.05). The cell cycle showed that the proportion of G0 and G1 increased, but S, G2-M phases obviously decreased (P<0.05). Compared with the tumor-bearing group, the neutrophils increased in the tripterine group, and the expression of early promyelocyte, NB4 cell, B lymphocyte, leukocyte, P-selectin, sVCAM-1 and sICAM-1 protein decreased (P<0.05), moreover, the ratio of G0 and G1 decreased, the G2-M and S phases increased (P<0.05). Among which sVCAM-1 and sICAM-1 of leucocyte, NB4 cell, P-selectin and adhesion molecules in tripterine group showed significant difference as compared with arsenic trioxide group (P<0.05). Tripterine may not only inhibit the expression of sVCAM-1 and sICAM-1 proteins in APL tumor-bearing mice and reduce the adhesion of tumor cells, but also block tumor cells at G2-M and S phases, thereby exerting therapeutic effect on APL.

Pharmacokinetic-Pharmacodynamic Modeling of Tumor Targeted Drug Delivery Using Nano-Engineered Mesenchymal Stem Cells.

Nano-engineered mesenchymal stem cells (nano-MSCs) are promising targeted drug delivery platforms for treating solid tumors. MSCs engineered with paclitaxel (PTX) loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) are efficacious in treating lung and ovarian tumors in mouse models. The quantitative description of pharmacokinetics (PK) and pharmacodynamics (PD) of nano-MSCs is crucial for optimizing their therapeutic efficacy and clinical translatability. However, successful translation of nano-MSCs is challenging due to their complex composition and physiological mechanisms regulating their pharmacokinetic-pharmacodynamic relationship (PK–PD). Therefore, in this study, a mechanism-based preclinical PK–PD model was developed to characterize the PK–PD relationship of nano-MSCs in orthotopic A549 human lung tumors in SCID Beige mice. The developed model leveraged literature information on diffusivity and permeability of PTX and PLGA NPs, PTX release from PLGA NPs, exocytosis of NPs from MSCs as well as PK and PD profiles of nano-MSCs from previous in vitro and in vivo studies. The developed PK–PD model closely captured the reported tumor growth in animals receiving no treatment, PTX solution, PTX-PLGA NPs and nano-MSCs. Model simulations suggest that increasing the dosage of nano-MSCs and/or reducing the rate of PTX-PLGA NPs exocytosis from MSCs could result in improved anti-tumor efficacy in preclinical settings.

Postweaning Grouping as a Strategy to Reduce Singly Housed Male Mice.

Rearing laboratory mice in groups is important since social isolation after weaning induces brain alterations, which entails behavioral abnormalities in adulthood. Age is an important factor when grouping males of different litters due to inter-male aggressiveness. The aim of this study was to determine whether newly weaned mice could safely be grouped with late juvenile or early and late pubescent mice, and whether cage cleaning, the number of the hosting group members and testosterone plasma levels have any influence. Newly weaned C57BL/6J, CD1, and SCID Beige male mice were systematically grouped with same strain late juvenile, early or late pubescent male mice in clean or dirty cages of 1, 2 or 3 hosting members. We also analyzed plasma testosterone levels at different postnatal days. Our result showed that only strain and hosting male's age influence agonistic behavior toward newly weaned mice. Thus, in order not to house a recently weaned male alone, we would recommend grouping it with late juvenile same strain mice in all studied strains. In the same way, CD1 and SCID Beige pubescent mice will admit a newly weaned mouse in their group. However, we would not recommend grouping newly weaned and pubescent C57BL/6J males.

Sclerostin-Neutralizing Antibody Treatment Rescues Negative Effects of Rosiglitazone on Mouse Bone Parameters.

Obesity, a growing pandemic, is a risk factor for many cancers and causes increased bone marrow adipose tissue (BMAT). in vitro studies and obese animal models suggest that BMAT contributes to cancer progression, but there is a lack of preclinical models to directly test BMAT's role in cancer. Overactivation of peroxisome-proliferator-activated receptor-γ (PPARγ) can skew bone formation and resorption rates, resulting in increased BMAT and trabecular bone loss. Thiazolidinediones (eg, rosiglitazone) are anti-diabetic therapies that promote adipogenesis through PPARγ activation. We investigated if rosiglitazone increases BMAT in an immunocompromised model, commonly used in cancer research, and if these effects could be reversed by co-administering a bone anabolic agent (sclerostin-neutralizing antibody [Scl-Ab]), which has been shown to inhibit adipogenesis, using DXA, μCT, OsO4 μCT, and dynamic histomorphometry. Four weeks of rosiglitazone in female SCID Beige mice (cohort 1) significantly decreased trabecular bone volume (BV/TV) by about one-half, through increased osteoclast and suppressed osteoblast activity, and significantly increased BMAT. In cohort 2, mice were administered rosiglitazone ± Scl-Ab for 4 weeks, and then rosiglitazone was discontinued and Scl-Ab or vehicle were continued for 6 weeks. Scl-Ab significantly increased bone parameters (eg, BV/TV, N.Ob/B.Pm, and MS/BS) in both groups. Scl-Ab also overcame many negative effects of rosiglitazone (eg, effects on trabecular bone parameters, increased mineralization lag time [MLT], and decreased bone formation rate [BFR]). Interestingly, Scl-Ab significantly decreased rosiglitazone-induced BMAT in the femur, mostly due to a reduction in adipocyte size, but had a much weaker effect on tibial BMAT. These data suggest targeting sclerostin can prevent rosiglitazone-induced bone loss and reduce BM adiposity, in some, but not all BMAT locations. Collectively, our data demonstrate that rosiglitazone increases BMAT in SCID Beige mice, but concomitant changes in bone may confound its use to specifically determine BMAT's role in tumor models. © 2020 American Society for Bone and Mineral Research (ASBMR).

Sirt6 promotes tumorigenesis and drug resistance of diffuse large B-cell lymphoma by mediating PI3K/Akt signaling

BackgroundSirtuin 6 (Sirt6) is a highly conserved ADP-ribosylase and NAD+ dependent deacylase, involved in broad cellular processes. This molecule possesses contradictory roles in carcinogenesis, as it has been documented to both suppressing and augmenting tumor growth. This project aimed to explore the expression and functions of Sirt6 in diffuse large B-cell lymphoma (DLBCL), especially with regards to the regulatory role of OSS_128167, a novel small molecular inhibitor targeting Sirt6.MethodsImmunohistochemistry (IHC) was conducted to assess the expression of Sirt6 on paraffin-embedded tissues. Microarray dataset GSE32918 and GSE83632 were obtained from Gene Expression Omnibus and survival analysis was performed. Lentivirus vectors either encoding shSirt6, lvSirt6 or empty lentiviral vector were stably transfected into DLBCL cells. LY1 cell transfected with shSirt6 were performed RNA-sequencing (RNA-seq) analysis, functional enrichment analyses of gene ontology (GO) and gene set enrichment analysis (GSEA). DLBCL cells were subcutaneously injected to SCID beige mice to establish xenograft models.ResultsSirt6 is found to be overexpressed in DLBCL, and is related to poor prognosis. Sirt6-deprived DLBCL cells displayed augmented sensitivity towards chemotherapy, higher rates of apoptosis, dysfunctional cell proliferation, and arrested cell cycle progression between the G2 and M phases. Selective OSS_128167-mediated Sirt6 blockage resulted in similar anti-lymphoma effects when compared to Sirt6 knocked-down DLBCL cells. PI3K signaling along with phosphorylation of its downstream targets was reduced upon Sirt6 downregulation. Xenograft models subjected to either OSS_128167 treatment or Sirt6-knockdown showed suppressed tumor growth and lower Ki-67 level.ConclusionsThese findings provide mechanistic insights into the oncogenic activity of Sirt6 in DLBCL for the first time and highlighted the potency of OSS_128167 for novel therapeutic strategies in DLBCL.

Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

The impressive successes of immune checkpoint blockade antibodies to treat various types of cancer are limited to minor subsets of patients. Combination therapy strategies, including with chemotherapy, are being explored to possibly improve the efficacy of immunotherapies. Here we report results regarding the use of an immunostimulatory regimen of metronomic cyclophosphamide (CTX). We show that in orthotopic models of syngeneic murine triple-negative breast cancer (EMT6), CTX administered at 140 mg/kg every 6 days (CTX140 1q6d) is superior at inhibiting primary tumor growth when compared to maximum tolerated dose or daily oral (continuous) low-dose CTX. In SCID or SCID beige mice, anti-tumor effects of CTX140 1q6d are reduced, reinforcing the therapeutic contribution of the adaptive and innate immune systems. In a second breast cancer model (SP1-AC2M2), CTX140 1q6d again showed clear superiority in anti-tumor effects, causing complete tumor regressions; however, these mice were not protected from subsequent tumor re-challenge, suggesting absence of immune memory. We also show that in an aggressive and metastatic cisplatin-resistant variant (EMT6-CDDP), CTX140 1q6d is superior and invokes an influx of intra-tumoral CD4+ and CD8+ T cells. CTX increases expression of tumor cell PD-L1; however, when combined with concomitant PD-L1 antibody therapy none of the CTX regimens showed increased benefit. This work sheds light on the potential use of metronomic CTX for the treatment of breast cancer, in particular using the quasi-weekly regimen, but also underscores the complexity of the anti-tumor mechanisms and potential to improve immune checkpoint therapy efficacy.