SCID Mouse Xenograft Model Research Articles

Abstract 6296: Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload

Abstract C-type lectin domain family 12 member A (CLEC12A, CLL-1) is a single-pass transmembrane protein of 265 amino acids that is found on monocytes and AML blasts. We have created and characterized antibody-drug conjugates (ADCs) based on a humanized CLEC12A specific antibody to determine whether CLEC12A may be exploited as a therapeutic target for AML. The payload is a proprietary PBD prodrug with a beta-glucuronide trigger, and the linker-payload was conjugated through farnesyltransferase-mediated functionalization. The ADC demonstrated sub-nM cytotoxicity in vitro against several CLEC12A-positive cell lines including HL-60 and PL21. Leu234Ala/Leu235Ala (LALA) mutations were employed to reduce Fc gamma receptor binding and to avoid on-target toxicity. The LALA mutation-bearing ADC displayed a nearly 50-fold decrease in cytotoxicity towards CD34+ hematopoietic stem cells. Its potency was reduced but still sufficient with IC50 values of 61 pM and 15 pM against HL60 and PL21, respectively. Regardless of LALA mutation, the ADCs demonstrated potent antitumor activities with a complete regression at a dose of 0.5 mpk against a subcutaneous HL-60 SCID mouse xenograft model. When the LALA-mutated ADC was tested against a disseminated NSGA mouse model of HL-60-luc, all treated animals survived without clinical symptoms for three weeks after treatment, whereas vehicle-treated animals exhibited morbidity 19 days after treatment or 35 days after tumor implantation. The bone marrow of ADC-treated animals appeared to have nearly fully recovered, whereas that of vehicle-treated animals showed necrosis or tumor growth. In cynomolgus monkeys, the LALA-mutated ADC had a half-life of 82 hours at a dose of 0.2 mpk, and target-mediated drug disposition appeared weak or negligible. Our preclinical studies have shown that CLEC12A targeting ADC can be used as a therapeutics for treating AML. Citation Format: Jinwon Jung, Juhee Kim, Bora Lee, Jung A Kwon, Suyoun Lee, Byeongmin Yoo, Minji Ko, Ilhwan Ryu, Donghoon Yeom, Kyoungjae Lee, Jaehyun Eom, Hanbyul Lee, Jinhyung Ahn, Eunsil Sung, Weonkyoo You, Sang Hoon Lee, Myeong Joo Kim, Keon Woo Kwon, Hyun Joo Bae, Yun-Hee Park, Ho Young Song, Chul-Woong Chung. Preclinical study of a novel anti-CLEC12A antibody-drug conjugate with a glucuronide-protected pyrrolobenzodiazepine payload [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 6296.

Abstract 1621: L105, a next generation of Bcl-2 inhibitor, overcomes Bcl-2 mutation and exhibits superior antitumor activity

Abstract Background: The Bcl-2 inhibitor venetoclax is approved for the treatment of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). However, disease progression occurs when Ventoclax long-term treatment as a single agent driven by the recurrent Bcl-2 mutations, such as G101V, D103Y, D103E, et al. Here, we described the discovery of a next generation Bcl-2 inhibitor L105 with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and venetoclax resistant hematological cancers. Methods: Biochemical inhibition assays were conducted by TR-FRET based assay. Proliferation inhibition assays on a panel of hematologic tumor cells were carried out by MTS assay. Caspase-3 activation in RS4;11 was determined by western blot. Preclinical pharmacokinetics studies were assayed in mice and dogs and parameters were determined by LC-MS. In vivo pharmacodynamic studies were performed in RS4;11 and OCI-LY10 derived xenograft SCID mouse models. In vitro human platelet toxicity was measured by CellTiter-Glo assay. Cytochrome P450 inhibition was measured in fluorometric assay. Results: In biochemical assays, the IC50 of L105 against Bcl-2 WT, Bcl-2 G101V, Bcl-2 D103Y, Bcl-2 D103E with IC50 of 0.42, 0.83, 0.97 and 5.78 nM respectively,while those of venetoclax were 1.89, 704.27, 470.19 and 243.30 nM respectively. In cellular assays, the IC50 of L105 in RS4;11, RS4;11 overexpressing Bcl-2 G101V, OCI-LY10, MV4-11, Mino, Toledo, Maver-1, SU-DHL-6 and REC-1 were 0.30-243.36 nM, while those of venetoclax were 2.91-3360.79 nM. L105 showed >2000 folds Bcl-2 selectivity over Bcl-xL. Furthermore, in vitro platelet viability assay showed L105 induced hypotoxicity at similar level of venetoclax. In pharmacodynamics studies, 5mpk L105 induced more rapid and robust antitumor effect in RS4;11 xenograft than venetoclax at 10 mpk did, and 15mpk L105 showed significant longer and stronger tumor suppression response than venetoclax at 25 mpk did. In the preclinical pharmacokinetic (PK) studies, L105 showed excellent PK properties in ICR mice and beagle dogs, in terms of long T1/2 and high dose-normalized AUC. L105 also exhibited a less drug-drug interaction potential, as no evidence on CYPs inhibition (IC50 >20 μM), while venetoclax potently inhibited CYP2C9 with IC50 at 1.12 μM. In 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mpk, and only leukopenia was observed at similar level of venetoclax. Conclusions: L105 showed great potency on wild type and acquisition resistance mutations of Bcl-2, excellent oral bioavailability, and superior preclinical anti-tumor activities and safety profiles. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Citation Format: Xingguo Liu, Yang Lou, Yizhe Wu, Mingbo Su, Anhui Gao, Jinglai Huang, Li Zhong, Xinglu Zhou. L105, a next generation of Bcl-2 inhibitor, overcomes Bcl-2 mutation and exhibits superior antitumor activity [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 1621.

Epibrassinolide impaired colon tumor progression and induced autophagy in SCID mouse xenograft model via acting on cell cycle progression without affecting endoplasmic reticulum stress observed in vitro.

Epibrassinolide is a member of brassinosteroids with a polyhydroxysteroid structure similar to steroid hormones of vertebrates. It was shown that EBR decreased cell proliferation and induced apoptosis in different colon cancer cell lines without exerting a cytotoxic effect in epithelial fetal human colon cells. This finding highlighted the potential of epibrassinolide in clinical therapeutic setup. In our previous studies, we showed that epibrassinolide was able to induce apoptosis via endoplasmic reticulum stress. Recently, we also showed that endoplasmic reticulum and apoptotic stresses can be prevented via autophagic induction in non-cancerous epithelial or aggressive forms of cancer cells. Therefore, here in this study, we evaluated the anti-tumoral effect of epibrassinolide as well as the autophagy involvement in the aggressive forms of colon cancer cell lines as well as in vivo SCID mouse xenograft colon cancer model for the first time. For this purpose, SCID mouse model was used for subcutaneous injection of colon cancer cells in matrigel formulation. We found that autophagy is induced in both in vitro and in vivo models. Following tumor formation, SCID mice were treated daily with increasing concentrations of epibrassinolide for two weeks. Our findings showed that EBR inhibited the volume and diameter of the tumor in a dose-dependent manner by causing cell cycle arrest. Therefore our data suggest that epibrassinolide exerts a cytostatic effect on the agrressive form of colon cancer model in vivo, without affecting endoplasmic reticulum stress and the induction of autophagy might have role in this effect of epibrassinolide.

Discovery of HZ-L105, a Next Generation of Bcl-2 Inhibitor, Overcomes Bcl-2 Mutation and Exhibits Superior Antitumor Activity

Introduction: Bcl-2, a key regulator of apoptosis pathway, is over-expressed in many hematologic malignancies, which results in blocking of apoptosis and cell survival. Targeting Bcl-2 for the treatment of malignant hematological tumors has been well validated and demonstrated by FDA approval of the first generation Bcl-2 inhibitor Venetoclax for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and acute myeloid leukemia (AML). Although Venetoclax provides significant clinical benefits, there are reports show that after long-term monotherapy patients develop disease progression , often driven by acquisition of Bcl-2 Gly101Val (G101V), Asp103Tyr (D103Y) resistance mutations. Here, we described the discovery of a next generation Bcl-2 inhibitor HZ-L105 as with high potency against Bcl-2, Bcl-2(G101V) and Bcl-2(D103Y) for treatment of Bcl-2 dependent and Venetoclax-resistant hematological cancers. Methods:Biochemical inhibition assays were conducted towards Bcl-2/Bcl-2 G101V/Bcl-2 D103Y/Bcl-xL by time-resolved fluorescence resonance energy transfer (TR-FRET);Cellular proliferation tests of RS4;11 and RS4;11-G101V overexpressed stable line were carried out using MTS assay;Human platelet viability was measured by Cell-titer Glo assay;Cytochrome P450 inhibition assays were performed using fluorometric assay;In vivo pharmacodynamic (PD) studies were performed in RS4;11-derived subcutaneous xenograft SCID mouse model;Preclinical pharmacokinetics (PK) studies were assayed in mice and dogs and parameters were determined by LC-MS. Results: In biochemical assays, HZ-L105 potently inhibited Bcl-2 WT, Bcl-2 G101V and Bcl-2 D103Y with IC50 of 0.54 nM, 6.16 nM and 5.57 nM，while Venetoclax's IC50 of 1.62 nM, 440.6 nM and 279.8 nM, respectively. Therefore, it is expected to overcome the acquisition of Bcl-2 resistance mutations. In cellular assays, HZ-L105 suppressed cell growth not only in RS4;11 but also in RS4;11-G101V stable line (IC50=0.30 nM in RS4;11 and 243.36 nM in RS4;11-G101V), which was much more superior to Venetoclax (IC50= 2.91 nM in RS4;11 and 3360.79 nM in RS4;11-G101V). HZ-L105 was also highly selective against Bcl-2, showing >1000 folds selectivity to Bcl-xL. Furthermore, platelet viability assay in vitro showed HZ-L105 induced hypotoxicity as similarly to Venetoclax, suggesting its good potential to avoid thrombocytopenia due to Bcl-xL inhibition. In the preclinical Pharmacodynamics studies, oral administration of HZ-L105 induced rapid and robust antitumor effect in RS4;11 ALL xenografts. HZ-L105 at a dose of 5 mg/kg demonstrated significantly excellent efficacy than Venetoclax at a dose 15mg/kg in RS4;11-derived xenograft mouse model. In the preclinical pharmacokinetic studies, HZ-L105 showed excellent pharmacokinetic properties in ICR mice and beagle dogs, with long T1/2 and high dose-normalized AUC. HZ-L105 did not reduced hERG tail current (% current blocked 2.87% at 30μM), indicating the compound has a low potential to induce QT prolongation. HZ-L105 has less drug-drug interaction potential, as evidenced by no inhibitory effect on CYPs, especially in CYP2C9 (IC50 >20 μM) compared with Venetoclax (IC50=1.12 μM). Besides, in 14-days oral gavage toxicity study in mice, no compound related deaths occurred at 15, 50 and 300 mg/kg/day, and only leukopenia was observed similarly to Venetoclax. Conclusion: In summary, HZ-L105 as a next generation Bcl-2 inhibitor, showed great potency on wild type and acquisition resistance mutations of Bcl-2 in vitro, excellent oral bioavailability, superior anti-tumor activities on preclinical xenograft model, and safety profiles,. It may provide new thoughts on treatment for a wide range of Bcl-2-dependent and Venetoclax-resistant hematological cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P-110: Activity and tolerability of lenalidomide (LLD) is enhanced following low-dose continuous percutaneous delivery compared to once daily dosing in an NCIH929 MM Xenograft model in SCID mice

P-110: Activity and tolerability of lenalidomide (LLD) is enhanced following low-dose continuous percutaneous delivery compared to once daily dosing in an NCIH929 MM Xenograft model in SCID mice

Anti-tumor Effect of Folate-Binding Protein: In Vitro and In Vivo Studies.

Folate receptor (FR) overexpression in a wide range of solid tumors provides an opportunity to develop novel, targeted cancer therapeutics. In this study, we investigated whether prebinding the chemotherapeutic methotrexate (MTX) to folate-binding protein (FBP), the soluble form of FR, would enable the protein to serve as a targeted therapeutic vector, enhancing uptake into tumor cells and improving therapeutic efficacy. In an in vivo study, using an FR-overexpressing KB xenograft model in SCID mice, modest improvement in inhibiting tumor growth was observed for the MTX/FBP mixtures as compared to saline control and free MTX. Surprisingly, FBP alone inhibited tumor growth compared to saline control, free MTX, and FBP/MTX. In order to better understand this effect, we investigated the cytotoxicity of micromolar concentrations of FBP in vitro using the KB, HeLa, and A549 cancer cell lines. Our results revealed concentration-dependent apoptosis (24 h; 10-50 μM) in all three cell lines accompanied by a time- and concentration-dependent reduction (6, 12, and 24 h; 10-50 μM) in metabolic activity and compromised cell plasma membrane integrity. This study demonstrates an apoptosis pathway for cytotoxicity of FBP, an endogenous serum protein, in cancer cell lines with widely varying levels of FR expression. Furthermore, in vivo tumor growth suppression for xenograft KB tumors in SCID mice was observed. These studies suggest novel strategies for the elimination of cancer cells employing endogenous, serum transport proteins.

Aurora Kinase Inhibition Overcomes Primary Venetoclax Failure and Leads to Synthetic Lethality in BCL2-Positive Lymphomas Via Upregulation of P53/P21/BAX Axis

Aurora Kinase Inhibition Overcomes Primary Venetoclax Failure and Leads to Synthetic Lethality in BCL2-Positive Lymphomas Via Upregulation of P53/P21/BAX Axis

Abstract 2887: Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia

Abstract Phenotyping of AML patient samples shows that CD33, a myeloid cell marker, and CD7, a lymphoid cell marker, are specifically co-expressed in 25% of AML cases. Development of a bispecific antibody drug conjugate (ADC) dependent on co-expression of CD7 and CD33 may lead to an ADC with better tumor-cell selectivity than the anti-CD33 ADC, gemtuzumab ozogamicin. Anti-CD7 and -CD33 Fabs were expressed in HEK293 cells, and affinities assessed using surface plasmon resonance. Chemically cross-linked anti-CD7, anti-CD33 Bi-Fabs were produced using click chemistry via an individual cysteine residue in each CH1 domain. Bi-Fabs were conjugated to valine-citrulline-monomethylauristatin E (vcMMAE) and maleimidocaproyl monomethylauristatin F (mcMMAF) at native cysteine residues. Bi-Fab drug conjugates were assessed for cytotoxicity against HNT-34 and Kasumi-3 cells (CD7+CD33+), and assessed for selectivity against primary human T cells and myeloid progenitor cells, using monospecific Bi-Fab and gemtuzumab drug conjugates with equivalent drug-antibody ratios as controls. The activity of the lead Bi-Fab, BVX130-mcMMAF, was assessed in a subcutaneous (s.c.) xenograft model in SCID mice implanted with HNT-34 cells. Animals (n=10/group) received BVX-130-mcMMAF (10mg/kg twice weekly i.v. for 4 weeks), cytarabine at its maximum tolerated dose (20mg/kg, 5 days on 2 days off i.p. for 3 weeks), or vehicle control for 4 weeks. The wild-type Fabs had Kd values of 3.6nM (anti-CD7 Fab) and 1.6 nM (anti-CD33 Fab) for their respective antigens. The resulting anti-CD7, anti-CD33 Bi-Fab MMAE conjugate (BVX100-vcMMAE) was highly cytotoxic to HNT-34 and Kasumi-3 cells with IC50 values of 0.20±0.04nM and 0.20±0.03nM respectively, compared to IC50 values of 1.3nM and 1.2nM for gemtuzumab MMAE. BVX100-vcMMAE had 5-fold lower cytotoxicity towards Jurkat cells (CD7+CD33−) and >10-fold margins relative to MV4.11 and SHI-1 (CD7−CD33+) cells whereas corresponding homo-dimeric Bi-Fabs were non-selective. Affinity reduction of the CD7 Fab resulted in Bi-Fab drug conjugates with similar activity and improved selectivity. One such Bi-Fab, BVX130mcMMAF, had no measurable cytotoxicity to resting T-cells, and did not inhibit colony formation in a GM-CFU assay, at concentrations up to 10nM. Treatment of mice carrying s.c. HNT-34 tumors with BVX130-mcMMAF resulted in Tumor Growth Inhibition (TGI) values of 100% (98% regression, p<0.001) and 100% (97% regression, p<0.001) at days 21 and 28 respectively, with three animals tumor-free at the end of dosing. Treatment with cytarabine resulted in TGI of 100% (47% regression, p<0.001) at day 21. No adverse effects were observed in the BVX130-mcMMAF treatment group. Anti-CD7, anti-CD33 Bi-Fab drug conjugates are highly cytotoxic to CD7+CD33+ cells in vitro and in vivo, and have substantially greater selectivity than monospecific drug conjugates for such cells, supporting the development of bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of CD7+/CD33+ AML. Citation Format: Richard Bethell, Cath Eberlein, Victoria Pollard, Theonie Georgiou, George Orphanides, Lorraine Mooney, Jane Kendrew, Tiffany Daniels. Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2887.

An Anti-PSMA Immunotoxin Reduces Mcl-1 and Bcl2A1 and Specifically Induces in Combination with the BAD-Like BH3 Mimetic ABT-737 Apoptosis in Prostate Cancer Cells.

Background: Upregulation of anti-apoptotic Bcl-2 proteins in advanced prostate cancer leads to therapeutic resistance by prevention of cell death. New therapeutic approaches aim to target the Bcl-2 proteins for the restoration of apoptosis. Methods: The immunotoxin hD7-1(VL-VH)-PE40 specifically binds to the prostate specific membrane antigen (PSMA) on prostate cancer cells and inhibits protein biosynthesis. It was tested with respect to its effects on the expression of anti-apoptotic Bcl-2 proteins. Combination with the BAD-like mimetic ABT-737 was examined on prostate cancer cells and 3D spheroids and in view of tumor growth and survival in the prostate cancer SCID mouse xenograft model. Results: The immunotoxin led to a specific inhibition of Mcl-1 and Bcl2A1 expression in PSMA expressing target cells. Its combination with ABT-737, which inhibits Bcl-2, Bcl-xl, and Bcl-w, led to an induction of the intrinsic apoptotic pathway and to a synergistic cytotoxicity in prostate cancer cells and 3D spheroids. Furthermore, combination therapy led to a significantly prolonged survival of mice bearing prostate cancer xenografts based on an inhibition of tumor growth. Conclusion: The combination therapy of anti-PSMA immunotoxin plus ABT-737 represents the first tumor-specific therapeutic approach on the level of Bcl-2 proteins for the induction of apoptosis in prostate cancer.

Anti-Cancer Effects of CKD-581, a Potent Histone Deacetylase Inhibitor against Diffuse Large B-Cell Lymphoma.

Double-hit lymphoma (DHL) and double-expressor lymphoma (DEL) are aggressive forms of lymphoma that require better treatments to improve patient outcomes. CKD-581 is a new histone deacetylase (HDAC) inhibitor that exhibited a better safety profile in clinical trials compared to other HDAC inhibitors. Here, we demonstrate that CKD-581 inhibited the class I–II HDAC family via histone H3 and tubulin acetylation. CKD-581 treatment also up-regulated the phosphorylation of histone H2AX (γH2AX, DNA double-strand break marker), and reduced levels of MYC and anti-apoptotic proteins such as BCL-2, BCL-6, BCL-XL, and MCL-1 in DH/DE-diffuse large B cell lymphoma (DLBCL) cell lines. Ultimately, CKD-581 also induced apoptosis via poly(ADP ribose) polymerase 1 (PARP1) cleavage. In a DLBCL SCID mouse xenograft model, CKD-581 exhibited anti-cancer effects comparable with those of rituximab (CD20 mAb). Our findings suggest that CKD-581 could be a good candidate for the treatment of DLBCL.

Abstract B14: Natural killer immune response is promoted by the treatment of B-cell lymphoma cancer cells with membrane ionophores

Abstract Introduction: Over the last two decades, very compelling clinical and experimental evidences have accelerated the use of natural killer (NK) cells’ properties for the recognition and eradication of hematologic malignancies. NK cells mediate antitumor killing responses via antibody-dependent cell-mediated cytotoxicity (ADCC). In order to accelerate and sustain NK antitumor immunity, antibodies targeting antigens expressed on the membrane surface of cancer cells are used as therapeutic interventions. Here, we investigated the antitumor benefit of the combination of the therapeutic anti-CD20 antibody, rituximab, with shortlisted membrane ionophores to overcome the resistance of B-cell lymphomas toward anti-CD20 targeted monoclonal antibodies. Methods: Flow cytometry was used to quantify cell surface protein levels and the rate of tumor cell death. Serum and PBMCs of human healthy donors were used as source of complement and natural killer cells, respectively. ADCC, complement-dependent cytotoxicity (CDC), and degranulation assays were used to assess the sensitivity of NHL cell lines and primary cancer cells upon treatment with ionophores plus rituximab. RNA-seq and qPCR were used to identify and confirm the deferentially expressed genes upon ionophores treatment. GSEA, an online resource of the Broad Institute (Boston, MA), was used to screen the differentially expressed genes and major signaling pathways. Results: Our data show that the sublethal doses of these ionophores (< 0.5uM) greatly increased surface CD20 (protein target for rituximab therapy) levels onB-cell lymphoma (Burkitt and DLBCL) cell lines. B-cell lymphoma cell death induced by rituximab, either mediated by complement or NK cell cytotoxicities, was significantly increased upon treatment with ionophores. These in vitro effects have been confirmed in xenograft model in SCID mice. While rituximab treatment alone slightly delayed tumor growth, the combination of rituximab with ionophores caused a dramatic decrease (up to 3x) of tumor size. The anticipated signaling pathways influencing NK cell activity are currently being interrogated; transcriptomic analysis combined with GSEA will allow to identify markedly altered pathways that are potentially closely involved in enhancing the sensitivity of rituximab-treated B-cell lymphoma cells. Summary: The data indicate that ionophores are promising therapeutic agents that could strongly enhance CD20 expression on the surface of target tumor cells. When combined with therapeutic anti-CD20 monoclonal antibodies, impressive high NK cell-mediated cytotoxicity is demonstrated that potentially offsets the B lymphoma resistance to R-CHOP regimen. These results also support the clinical development of these molecules as adjuvant to existing cancer immunotherapies. Support: National Science Centre (NCN, Poland), ID: 2016/23/B/NZ5/02622; Ministry of Science and Higher Education in Poland, ID: DI2014007344 and iONKO grant. Citation Format: Abdessamad Zerrouqi, Anna Torun, Nina Miazek, Zofia Pilch, Jakub Golab, Beata Pyrzynska. Natural killer immune response is promoted by the treatment of B-cell lymphoma cancer cells with membrane ionophores [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B14.

Inhibition of growth of Asian keloid cells with human umbilical cord Wharton\u2019s jelly stem cell-conditioned medium

BackgroundKeloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton’s jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs).MethodsHuman Wharton’s jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model.ResultsqRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice (n = 10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30 days.ConclusionshWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects

BackgroundDiffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL.MethodsPHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK–MNK–eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect.ResultsAs in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model.ConclusionOur data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.

Omipalisib (GSK458), a Dual an-PI3K/mTOR Inhibitor, Exhibits in Vitro and In Vivo activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Reports of recurrent genomic alterations in Burkitt lymphoma (BL) have identified multiple recurrent alterations that result in activation of PI3K highlighting the importance of the PI3K/AKT/mTOR pathway in Burkitt lymphomagenesis. In a cell line model of resistant BL, we have previously identified an increase of PI3K/AKT/mTOR pathway activation suggesting a role in therapy resistance. While inhibition of PI3K-delta using the isoform specific inhibitor idelalisib has demonstrated clinical activity in indolent lymphomas, limited single agent activity has been observed in more aggressive variants. Pre-clinical investigation of idelalisib in BL indicated similar somewhat limited in vitro activity with synergistic activity in combination with chemotherapy. Broader inhibition of both upstream PI3K and downstream mTOR may exhibit more significant anti-lymphoma activity.Objectives: Investigate the in vitro and in vivo activity of the dual pan-PI3K/mTOR inhibitor omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: Experiments were conducted in Raji, Raji 4RH (chemotherapy-rituximab resistant), Ramos, and Daudi BL cells. Cell viability following exposure to omipalisib +/- chemotherapy was analyzed using Cell-Titer Glo and Alamar blu assays. Induction of apoptosis was assessed by flow cytometry for Annexin V (AV)-propidium iodide (PI) staining. Downstream effects of omipalisib on PI3K/Akt/mTOR signaling were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using PI staining. Synergy of combination exposures was determined by calculation of the combination index (CI) using CalcuSyn software. In vivo activity was evaluated using disseminated Raji and subcutaneous Ramos SCID mouse xenograft models. The survival end point was hind limb paralysis in the disseminated model and tumor diameter >2cm in the subcutaneous model. Mice were treated with vehicle or omipalisib daily by oral gavage. Median survival was compared by Kaplan-Meier analysis.Results: Exposure of BL cells to omipalisib for 24-72 hours resulted in a dose- and time-dependent decrease in viable cells at nM concentrations (48h IC50 values: Raji=1.2uM, Raji 4RH=0.02uM, Ramos=0.01uM, Daudi=0.01uM) (Figure 1A). Marked induction of apoptosis occurred following 72h exposure to omipalisib primarily in chemosensitive cells with half-maximal effect noted at approximately 200nM, but requiring significantly higher concentrations to induce apoptosis in therapy resistant Raji 4RH cells (%AV positive at 200nM: Raji=40.7%, Raji 4RH=4.4%, Ramos=59.4% and Daudi=46.9%). Downstream of PI3K/Akt/mTOR, S6 and GSK3β showed reduced phosphorylation after 30 minute omipalisib exposure. G1 cell cycle arrest occurred in all cell lines following exposure to omipalisib for 72 hours; however, chemotherapy-resistant Raji 4RH cells arrested in G2/M at higher concentrations. BL cells exposed to omipalisib in combination with either doxorubicin or dexamethasone, exhibited synergistic anti-tumor activity (CI<0.9) with synergistic induction of apoptosis in therapy sensitive cells exposed to omipalisib and chemotherapy. NOD-SCID mice injected via tail vein with Raji-luc (provided by Dr. Mitchell Cairo) and treated with omipalisib demonstrated decreased luciferase signal compared to controls (Figure 1C) while mice with established subcutaneous Ramos xenografted tumors treated with omipalisib exhibited slower tumor progression compared to controls (Figure 1B), though with only modest prolongation of survival (median 28 vs 34 days, n=15/group, p<0.05).Conclusion: Dual PI3K-mTOR inhibitor omipalisib suppresses the PI3K/Akt/mTOR pathway leading to induction of apoptosis, impaired BL cell proliferation in vitro and in vivo and exhibits synergistic in vitro activity when combined with cytotoxic chemotherapy highlighting the relevance of PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 5621: FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies

Abstract For tumor specific co-stimulation via 4-1BB (CD137) a novel tumor-stroma targeted FAP-4-1BB ligand (FAP-4-1BBL) was designed composed of a trimeric split 4-1BBL, a tumor-stroma targeted 4B9 Fab moiety recognizing fibroblast activation protein (FAP) and a heterodimeric Fc-region devoid of FcgR binding but maintained FcRn binding. In presence of a TCR signal 1 either from a T cell receptor MHCI/peptide interaction or through TCR engagement by a T-cell bispecific antibody, FAP-4-1BBL provides co-stimulation to T cells strictly dependent on cross-linking by FAP-expressing fibroblasts. Similarly, FAP-4-1BBL can provide co-stimulation to NK cells with activated FcgRIIIa signaling. FAP-4-1BBL was tested as monotherapy and combined with CEA-TCB, an anti-CEA T-cell bispecific antibody, in the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in human stem cell engrafted (HSC) NSG mice in comparison to DP47-4-1BBL, an analogous untargeted 4-1BBL fusion protein. For use in syngeneic mouse models in immunocompetent mice muFAP-4-1BB, a murine surrogate made of a mu4-1BB agonistic surrogate antibody fused to the variable region of the FAP antibody 4B9, was used. muFAP-4-1BB was tested as monotherapy and combined with a murine specific CEA-TCB surrogate (muCEA-TCB) and the muPD-L1 specific surrogate antibody 6E11 in the s.c. colorectal MC38-CEA model in CEA transgenic (Tg) C57BL/6 mice. For combination with ADCC-mediating antibodies FAP-mu4-1BBL, a hybrid FAP-mu4-1BBL fusion protein, was generated. FAP-mu4-1BBL was tested as monotherapy and combined with the ADCC-mediating anti-HER2 antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. In the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in HSC-NSG mice FAP-4-1BBL resulted in combined anti-tumoral efficacy in combination with CEA-TCB, whereas the respective monotherapies as well as the combination with the untargeted DP47-4-1BBL did not improve anti-tumor efficacy. In the syngeneic s.c. MC38-CEA model in CEA Tg C56BL/6 mice, a model with natural FAP expression due to fibroblast infiltration, the muFAP-4-1BB surrogate antibody resulted in combined anti-tumor efficacy, both combined with muCEA-TCB and muPD-L1 antibodies including the induction of tumor remission. Finally, the hybrid FAP-mu4-1BBL surrogate resulted in improved anti-tumor efficacy combined with the ADCC-mediating antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. These data show that FAP-4-1BBL is a versatile combination partner for cancer immunotherapy mediating FAP-dependent co-stimulation to T and NK cells in combination with PD-L1 checkpoint inhibition, TCBs and ADCC-mediating antibodies. Clinical evaluation of this novel therapeutic approach is planned early 2018. Citation Format: Johannes Sam, Christina Claus, Claudia Ferrara, Sabine Lang, Valeria Nicolini, Sara Colombetti, Volker Teichgräber, Stefan Evers, Marina Bacac, Pablo Umana, Christian Klein. FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5621.

Abstract 5068: Distinct pro-inflammatory cytokines in prostate fibroblasts from African-Americans with prostate cancer increase the tumorigenicity of cancer cells

Abstract Introduction: African-American (AA) men are disproportionately affected by prostate cancer (PCa) compared with Caucasian (CA) men. AA men have higher PCa incidence and, once diagnosed, a higher stage-specific mortality rate (2.4 times higher) than CA men. Although socioeconomic factors partially explain such differences, a growing body of new scientific evidence suggests that biological factors, such as differences at the genetic and molecular level could be more critical than previously thought for the observed racial PCa disparities in incidence and outcome. Recent gene expression studies revealed that differences in the tumor microenvironment (TME) of AA vs. CA prostate tumors could affect the immune-inflammatory milieu. Fibroblasts are important regulators of stromal-epithelial paracrine interactions (SEI) in the TME. We isolated fibroblasts from AA and CA PCa patients and compared their biological effects in vitro and in vivo on PCa cell lines. Methods: Prostate fibroblasts were isolated from AA (PrF-AA) and CA (PrF-CA) patient tissues. Characterization of PrF included assaying their proliferation and expression of potential markers associated with the activation of carcinoma associated fibroblasts. In vitro effects of AA vs. CA fibroblasts on PCa cell proliferation and motility were studied. In vivo, the pro-tumorigenic properties of fibroblasts were tested using a subrenal xenograft model in SCID mice. RNA-Seq and cytokine array analysis of potential paracrine mediators of tumorigenesis was performed. Immunohistochemical analysis of a panel of putative stromal markers associated with racial differences was assessed in a cohort of PCa tissues from AA and CA. Results: Prostate fibroblasts from AA vs. CA men exhibit increased proliferation in response to known stromal mitogens. Expression of markers associated with myofibroblast activation (αSMA, vimentin and Tenascin-C) were significantly elevated in AA. Interestingly, expression of the androgen receptor (AR) and activation of AR signaling was significantly increased in PrF-AA compared to PrF-CA. The paracrine effects of PrF-AA on PCa cell proliferation and motility were significantly greater compared to those induced by PrF-CA. In an in vivo model, PRF-AA induced formation of larger and more invasive tumors by the AA PCa cell line E006AA compared to PRF-CA. Analysis of downstream regulatory pathways and potential paracrine mediators identified a panel of pro-inflammatory cytokines notably interleukins (IL6, IL11, IL17, IL18BP), growth factors (VEGF, FGF, BDNF), and other mediators enriched in PRF-AA. Conclusions: Prostate fibroblasts from African American men show enhanced secretion of pro-inflammatory and pro-proliferative mediators that can potentially increase the tumorigenicity of PCa cells through selective paracrine mechanisms. Citation Format: Marc Gillard, Alejandro Morales, Susan Crawford, Charles Brendler, Donald Vander Griend, Omar Franco. Distinct pro-inflammatory cytokines in prostate fibroblasts from African-Americans with prostate cancer increase the tumorigenicity of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5068.

Epigenetic reactivation of LINE-1 retrotransposon disrupts NuRD corepressor functions and induces oncogenic transformation in human bronchial epithelial cells.

Long interspersed nuclear element‐1 (LINE‐1 or L1) reactivation is linked to poor prognosis in non‐small‐cell lung carcinoma (NSCLC), but the molecular bases of this response remain largely unknown. In this report, we show that challenge of human bronchial epithelial cells (HBECs) with the lung carcinogen, benzo(a)pyrene (BaP), shifted the L1 promoter from a heterochromatic to euchromatic state through disassembly of the nucleosomal and remodeling deacetylase (NuRD) complex. Carcinogen challenge was also associated with partial displacement of constituent proteins from the nuclear to the cytoplasmic compartment. Disruption of NuRD corepression by genetic ablation or carcinogen treatment correlated with accumulation of L1 mRNA and proteins. Mi2β bound directly to the L1 promoter to effect retroelement silencing, and this response required the DNA‐ and ATPase‐binding domains of Mi2β. Sustained expression of L1 in HBECs was tumorigenic in a human–SCID mouse xenograft model, giving rise to tumors that regressed over time. Together, these results show that functional modulation of the NuRD constituent proteins is a critical molecular event in the activation of L1 retrotransposon. L1 expression creates a microenvironment in HBECs that is conducive to neoplasia and malignant transformation.

Effects of indole-3-carbinol on steroid hormone profile and tumor progression in a mice model of canine inflammatory mammarycancer

BackgroundIndole-3-carbinol, derived from Cruciferous vegetables is an estrogen receptor antagonist considered a preventive agent that is naturally present in diet. There are no previous studies on its effects in human inflammatory breast cancer or canine inflammatory mammary cancer that is the most aggressive type of breast cancer.MethodsThe aim of this study was to analyze the effect of indole-3-carbinol on a SCID mice xenograft model of canine inflammatory mammary cancer, using equivalent human oral dose as a preventive therapy in humans for 3 weeks.ResultsIndole-3-carbinol treatment decreased tumor proliferation and increased apoptosis, although tumor embolization and liver metastasis were observed in some animals. There was a characteristic subpopulation of lipid-rich cells and increased contents of select steroid hormones in tumor homogenates and serum.ConclusionsOur data reveal for the first time that the ingestion of indole-3-carbinol, as administered, diminishes proliferation and increases apoptosis of tumor cells in an experimental model of inflammatory breast cancer, although this effect could not be enough to avoid the appearance of tumor embolization and metastasis. Future clinical trials will be needed to clarify the usefulness of indole-3-carbinol in this cancer and to understand the molecular mechanisms involved.

Abstract A169: Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML

Abstract Background: Lysine-specific demethylase 1A (LSD1/KDM1A) is a flavin adenine dinucleotide (FAD)-dependent histone demethylase that specifically modifies histone H3 lysine 4 and lysine 9. LSD1 activity is implicated in the pathogenesis of several human cancers, and recent studies indicated that the inhibition of LSD1 is a promising therapeutic strategy for acute myeloid leukemia (AML). Decitabine and azacitidine, FDA-approved agents for the treatment of AML and myelodysplastic syndrome, alter global DNA methylation by inhibiting DNA methyltransferases (DNMTs), resulting in apoptosis and differentiation. Since it is reported that LSD1 directly demethylates and stabilizes DNMT1 by protection from degradation, we hypothesized that an LSD1 inhibitor could augment anti-AML activity of DNMT inhibitors. Previously, we demonstrated that TPC-144, a novel reversible LSD1 inhibitor, was efficacious in preclinical models of AML. Here, we report the activity of TPC-144 in combination with decitabine in cell-based and in vivo studies. Material and Methods: In vitro studies on growth inhibition and apoptosis were conducted using AML cell lines by measuring cellular ATP content (Cell-Titer Glo, Promega) and sub-G1 population, respectively. For expression changes of surface markers, cells were stained with specific antibodies (BD Pharmingen) and analyzed by flow cytometry. mRNA levels of LSD1 target genes were quantitated by real-time PCR. LINE-1 methylation levels were evaluated in Global DNA Methylation-LINE-1 Kit (Active Motif). In vivo efficacy was evaluated in human AML xenograft models in SCID mice. TPC-144 and decitabine was administered orally or intraperitoneally, respectively. Results: The combination of TPC-144 and decitabine showed synergistic growth inhibition of human AML cells in vitro. TPC-144 enhanced apoptosis induction in combination with low-dose decitabine at the concentrations at which TPC-144 decreased DNMT1 protein levels and LINE-1 methylation levels. Combination treatment strongly increased expression of surface markers of differentiation such as CD86 and CD11b, compared with single-agent treatment. In AML xenograft models, the combination of TPC-144 and decitabine was tolerated without signs of hematopoietic toxicities, and demonstrated curative antitumor efficacy compared with each inhibitor alone in a decitabine-sensitive xenograft model. Importantly, the combination treatment of TPC-144 and decitabine exhibited strong antitumor efficacy even in a decitabine-insensitive AML model. Conclusions: TPC-144, a novel potent and selective reversible LSD1 inhibitor, demonstrated synergistic antitumor efficacy in combination with decitabine in human AML cell lines and xenograft models. This combination was well tolerated and showed strong efficacy not only in decitabine-sensitive but also in decitabine-insensitive AML models. Therefore, combination of TPC-144 and decitabine could enhance their therapeutic value for the treatment of AML patients. Citation Format: Akiko Osada, Aki Kawagishi, Ryo Hatanaka, Takumitsu Machida, Kenjiro Ito, Satoshi Yamashita, Takahiro Ogawa, Tadashi Imaoka, Kenichi Matsuo, Teruhiro Utsugi, Yoshikazu Iwasawa. Combination of TPC-144, a reversible LSD1 inhibitor, and a hypomethylating agent resulted in synergistic antitumor efficacy in preclinical models of AML [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A169.

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates.

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.