Overcoming resistance to anabolic SARM therapy in experimental cancer cachexia with an HDAC inhibitor

No approved therapy exists for cancer-associated cachexia. The colon-26 mouse model of cancer cachexia mimics recent late-stage clinical failures of anabolic anti-cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx-024. The histone deacetylase inhibitor (HDACi) AR-42 exhibited anti-cachectic activity in this model. We explored combined SARM/AR-42 therapy as an improved anti-cachectic treatment paradigm. A reduced dose of AR-42 provided limited anti-cachectic benefits, but, in combination with GTx-024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR-42 suppressed the IL-6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx-024-mediated β-catenin target gene regulation was apparent in cachectic mice only when combined with AR-42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx-024 therapy and a blockade of GTx-024-mediated anabolic signaling. AR-42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx-024. Combining GTx-024, a clinically established anabolic therapy, with AR-42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.

Figure: Terminal tumor volume and fat mass in Study 2 Study 2, tumor‐bearing male mice receiving GTx‐024 (15 mg/kg; n = 10), AR‐42 (10 mg/kg; n = 9), combination (15 mg/kg GTx‐024 and 10 mg/kg AR‐42; n = 9) or vehicle (n = 7) and tumor‐free male mice receiving GTx‐024 (15 mg/kg; n = 6) or vehicle (n = 6) were treated daily by oral gavage for 12 days starting 6 days post‐injection of C‐26 cells.
A Terminal tumor volume comparisons between the initial (Study 1, day 18) and confirmatory (Study 2, day 17) combination studies. Statistics: P = 0.0009, Student’s t‐test of combined tumor volumes (Study 1 versus Study 2). ns, no significant differences among treatment groups within each study, one‐way ANOVA followed by Tukey’s multiple comparison test.
B Terminal epididymal fat pad mass.
Data information: V, G, A indicate statistically significant differences versus tumor‐bearing vehicle‐treated, tumor‐bearing GTx‐024‐treated, and tumor‐bearing AR‐42‐treated groups, respectively. P‐values are provided in Appendix Table S11, one‐way ANOVA followed by Tukey’s multiple comparison test. Data are presented as means ± SD.

Liva SG, Tseng YC, Dauki AM, Sovic MG, Vu T, Henderson SE, Kuo YC, Benedict JA, Zhang X, Remaily BC, Kulp SK, Campbell M, Bekaii-Saab T, Phelps MA, Chen CS, Coss CC. Overcoming resistance to anabolic SARM therapy in experimental cancer cachexia with an HDAC inhibitor. EMBO Mol Med. 2020 Feb 7;12(2):e9910. doi: 10.15252/emmm.201809910. Epub 2020 Jan 13. PMID: 31930715; PMCID: PMC7005646.

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Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression

Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.

Figure: AR-FL and AR-SVs expression in HCC primary samples and cell lines. (a, left) Top 100 (of 372) combined per-patient (x-axis) AR-FL (blue) and ligand-independent AR-SVs (red, as described in Supplementary Table 2). Numbers of patients with abundant AR-SV expression noted (inset) (a, right) RNA-Seq data from TCGA LIHC cohort were interrogated for AR-SVs transcript expression in female (n=121) and male (n=251). Statistical significance for AR-Svs expression in males vs females were evaluated using Mann-Whitney test **** p<0.0001 versus female. (b) Analyses of tumor RNA from 12 HCC majority cirrhotic and chronic hepatitis infected patients who underwent liver resection (male=10, female=2). Levels are compared to negative control THLE-2, normal liver cells, and positive control VCaP, PCa cells, to show abundant patient AR-FL and AR-v7 expression. Bars represent average technical duplicates and are matched for each patient. (c) Transcript abundance in transcript per million (TPM) of protein coding androgen receptor transcripts in 2 prostate cancer and 18 HCC cell lines from Cancer Cell Line Encyclopedia (CCLE) database. AR-FL (blue) and AR-SVs (red), as in Figure 1A, are presented. HCCLM3 cell data are not present in the CCLE. HCC cell AR transcript and protein expression were further validated by RT-PCR (d, h) and Western Blot (f, g), respectively. (d). RT-PCR analyses of AR-FL and AR-SVs transcripts in AR-positive prostate cancer (VCaP), AR-negative prostate cancer (DU145), AR-positive HCC (HCCLM3, SNU-423), AR-negative HCC (HepG2, PLC/PRF/5) and immortalized normal liver (THLE2) cell lines. (n=3, geometric mean ± SD). ARv4 and ARv12 were undetectable (supplementary Figure 5A). (e) Comparison of mean AR-FL and AR-v7 mRNA in primary samples as compared to the most abundant AR-SV expressing AR-positive HCC cells, HCCLM3, demonstrating robust AR isoform expression in primary HCC. (f) Western blot analysis with an N-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3 and SNU-423 cells and low molecular weight (MW) AR species in HCCLM3 cells migrating similarly to known AR-SVs in VCaP PCa cells. No AR-FL or lower MW species of AR were detected in HepG2, PLC/PRF/5, DU145, or THLE-2 cells. AR-negative HCC cell line, PLC/PRF/5, was transfected with either AR-FL expressing plasmid (PLC5_pAR-FL) or AR-v7 expressing plasmid (PLC5_pAR-v7) as positive controls for AR-FL and AR-v7, respectively. (g) Western blot analysis with a C-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3, SNU-423 and PLC5_pAR-FL cells. However, N-terminal directed monoclonal AR antibody detectable AR-SVs in VCaP and HCCLM3 cells are not detectable with c-terminal directed monoclonal AR antibody. WB performed with 35μg total protein lysate for all liver cell lines and 10μg for VCaP and DU145 and with primary N-terminal (CS#5153, Cell Signaling) or C-terminal AR mAb (ab52615, Abcam). (h). RT-PCR analyses of AR-FL and AR-SVs transcripts namely AR-v1, v3, and v7 in AR-positive HCC (HCCLM3, SNU-423, SNU475), AR-negative HCC (PLC/PRF/5) and immortalized normal liver (THLE2) cell lines (performed on low passage cells from ATCC Liver Cancer Panel TCP-1011, n=3, geometric mean ± SD). (i) Western blot analysis with an AR-v7 specific monoclonal AR antibody shows AR-v7 protein in 22Rv1, PLC5_pAR-v7, VCaP and SNU-475 cells. No AR-v7 reactive lower MW species of AR were detected in HCCLM3 cells. No AR-FL protein was detected in any of these cells. (j) To further confirm that the low molecular weight species that were detected by an AR-v7 specific AR mAb are C-terminal truncated splice variants, the blot presented in Figure 1I performed with a C-terminal targeting AR mAb was stripped, blocked and incubated with an N-terminal targeting AR mAb revealing abundant AR-FL in 22Rv1, VCaP and HCCLM3. The GAPDH blot from (i) is presented again here for convenience. No AR-FL isoform was detected in SNU-475 or PLC5_pAR-v7. However, low molecular weight AR species were detected in HCCLM3. (k) WGS of SNU-475 cells revealed a large ~48-kb hemizygous deletion in the AR-locus which included exons 4–8 of the AR-FL gene. This deletion is consistent with AR-v7 but not AR-FL expression and is strongly supported by sequencing data which included 56 read pairs with split reads and/or discordant pair alignments.
Figure: AR-FL and AR-SVs expression in HCC primary samples and cell lines. (a, left) Top 100 (of 372) combined per-patient (x-axis) AR-FL (blue) and ligand-independent AR-SVs (red, as described in Supplementary Table 2). Numbers of patients with abundant AR-SV expression noted (inset) (a, right) RNA-Seq data from TCGA LIHC cohort were interrogated for AR-SVs transcript expression in female (n=121) and male (n=251). Statistical significance for AR-Svs expression in males vs females were evaluated using Mann-Whitney test **** p<0.0001 versus female. (b) Analyses of tumor RNA from 12 HCC majority cirrhotic and chronic hepatitis infected patients who underwent liver resection (male=10, female=2). Levels are compared to negative control THLE-2, normal liver cells, and positive control VCaP, PCa cells, to show abundant patient AR-FL and AR-v7 expression. Bars represent average technical duplicates and are matched for each patient. (c) Transcript abundance in transcript per million (TPM) of protein coding androgen receptor transcripts in 2 prostate cancer and 18 HCC cell lines from Cancer Cell Line Encyclopedia (CCLE) database. AR-FL (blue) and AR-SVs (red), as in Figure 1A, are presented. HCCLM3 cell data are not present in the CCLE. HCC cell AR transcript and protein expression were further validated by RT-PCR (d, h) and Western Blot (f, g), respectively. (d). RT-PCR analyses of AR-FL and AR-SVs transcripts in AR-positive prostate cancer (VCaP), AR-negative prostate cancer (DU145), AR-positive HCC (HCCLM3, SNU-423), AR-negative HCC (HepG2, PLC/PRF/5) and immortalized normal liver (THLE2) cell lines. (n=3, geometric mean ± SD). ARv4 and ARv12 were undetectable (supplementary Figure 5A). (e) Comparison of mean AR-FL and AR-v7 mRNA in primary samples as compared to the most abundant AR-SV expressing AR-positive HCC cells, HCCLM3, demonstrating robust AR isoform expression in primary HCC. (f) Western blot analysis with an N-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3 and SNU-423 cells and low molecular weight (MW) AR species in HCCLM3 cells migrating similarly to known AR-SVs in VCaP PCa cells. No AR-FL or lower MW species of AR were detected in HepG2, PLC/PRF/5, DU145, or THLE-2 cells. AR-negative HCC cell line, PLC/PRF/5, was transfected with either AR-FL expressing plasmid (PLC5_pAR-FL) or AR-v7 expressing plasmid (PLC5_pAR-v7) as positive controls for AR-FL and AR-v7, respectively. (g) Western blot analysis with a C-terminal directed monoclonal AR antibody shows abundant AR-FL protein in HCCLM3, SNU-423 and PLC5_pAR-FL cells. However, N-terminal directed monoclonal AR antibody detectable AR-SVs in VCaP and HCCLM3 cells are not detectable with c-terminal directed monoclonal AR antibody. WB performed with 35μg total protein lysate for all liver cell lines and 10μg for VCaP and DU145 and with primary N-terminal (CS#5153, Cell Signaling) or C-terminal AR mAb (ab52615, Abcam). (h). RT-PCR analyses of AR-FL and AR-SVs transcripts namely AR-v1, v3, and v7 in AR-positive HCC (HCCLM3, SNU-423, SNU475), AR-negative HCC (PLC/PRF/5) and immortalized normal liver (THLE2) cell lines (performed on low passage cells from ATCC Liver Cancer Panel TCP-1011, n=3, geometric mean ± SD). (i) Western blot analysis with an AR-v7 specific monoclonal AR antibody shows AR-v7 protein in 22Rv1, PLC5_pAR-v7, VCaP and SNU-475 cells. No AR-v7 reactive lower MW species of AR were detected in HCCLM3 cells. No AR-FL protein was detected in any of these cells. (j) To further confirm that the low molecular weight species that were detected by an AR-v7 specific AR mAb are C-terminal truncated splice variants, the blot presented in Figure 1I performed with a C-terminal targeting AR mAb was stripped, blocked and incubated with an N-terminal targeting AR mAb revealing abundant AR-FL in 22Rv1, VCaP and HCCLM3. The GAPDH blot from (i) is presented again here for convenience. No AR-FL isoform was detected in SNU-475 or PLC5_pAR-v7. However, low molecular weight AR species were detected in HCCLM3. (k) WGS of SNU-475 cells revealed a large ~48-kb hemizygous deletion in the AR-locus which included exons 4–8 of the AR-FL gene. This deletion is consistent with AR-v7 but not AR-FL expression and is strongly supported by sequencing data which included 56 read pairs with split reads and/or discordant pair alignments.

Dauki AM, Blachly JS, Kautto EA, Ezzat S, Abdel-Rahman MH, Coss CC. Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression. Cancer Res. 2020 Feb 1;80(3):561-575. doi: 10.1158/0008-5472.CAN-19-1117. Epub 2019 Nov 4. PMID: 31685543; PMCID: PMC7002251.

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Population pharmacokinetics of lenalidomide in patients with B-cell malignancies

Aims: Lenalidomide is an immunomodulatory imide drug used broadly in the treatment of multiple myeloma and lymphoma. It continues to be evaluated in chronic lymphocytic leukaemia (CLL) at lower doses due to dose-related toxicities including tumour flare and tumour lysis syndrome. This study aimed to develop a population pharmacokinetic model for lenalidomide in multiple cancers, including CLL, to identify any disease-related differences in disposition.

Methods: Lenalidomide concentrations from 4 clinical trials were collated (1999 samples, 125 subjects), covering 4 cancers (multiple myeloma, CLL, acute myeloid leukaemia and acute lymphoblastic leukaemia) and a large dose range (2.5-75 mg). A population pharmacokinetic model was developed with NONMEM and patient demographics were tested as covariates.

Results: The data were best fitted by a 1-compartment kinetic model with absorption described by 7 transit compartments. Clearance and volume of distribution were allometrically scaled for fat-free mass. The population parameter estimates for apparent clearance, apparent volume of distribution and transit rate constant were 12 L/h (10.8-13.6), 68.8 L (61.8-76.3), and 13.5 h-1 (11.9-36.8) respectively. Patients with impaired renal function (creatinine clearance <30 mL/min) exhibited a 22% reduction in lenalidomide clearance compared to patients with creatinine clearance of 90 mL/min. Cancer type had no discernible effect on lenalidomide disposition.

Conclusions: This is the first report of a lenalidomide population pharmacokinetic model to evaluate lenalidomide pharmacokinetics in patients with CLL and compare its pharmacokinetics with other B-cell malignancies. As no differences in pharmacokinetics were found between the observed cancer-types, the unique toxicities observed in CLL may be due to disease-specific pharmacodynamics.

Figure: Plasma lenalidomide concentration–time profiles normalised for dose. The coloured dots represent each data point, with the colours corresponding to the dose ranges A, or cancer type B, according to the legend. ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; CLL, chronic lymphocytic leukaemia; MM, multiple myeloma.
Figure: Plasma lenalidomide concentration–time profiles normalised for dose. The coloured dots represent each data point, with the colours corresponding to the dose ranges A, or cancer type B, according to the legend. ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; CLL, chronic lymphocytic leukaemia; MM, multiple myeloma.

Hughes JH, Phelps MA, Upton RN, Reuter SE, Gao Y, Byrd JC, Grever MR, Hofmeister CC, Marcucci G, Blum W, Blum KA, Foster DJR. Population pharmacokinetics of lenalidomide in patients with B-cell malignancies. Br J Clin Pharmacol. 2019 May;85(5):924-934. doi: 10.1111/bcp.13873. Epub 2019 Feb 27. PMID: 30672004; PMCID: PMC6475687.

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Pharmacokinetic-Pharmacodynamic Model of Neutropenia in Patients With Myeloma Receiving High-Dose Melphalan for Autologous Stem Cell Transplant

High-dose melphalan (HDM) is part of the conditioning regimen in patients with multiple myeloma (MM) receiving autologous stem cell transplantation (ASCT). However, individual sensitivity to melphalan varies, and many patients experience severe toxicities. Prolonged severe neutropenia is one of the most severe toxicities and contributes to potentially life-threatening infections and failure of ASCT. Granulocyte-colony stimulating factor (G-CSF) is given to stimulate neutrophil proliferation after melphalan administration. The aim of this study was to develop a population pharmacokinetic/pharmacodynamic (PK/PD) model capable of predicting neutrophil kinetics in individual patients with MM undergoing ASCT with high-dose melphalan and G-CSF administration. The extended PK/PD model incorporated several covariates, including G-CSF regimen, stem cell dose, hematocrit, sex, creatinine clearance, p53 fold change, and race. The resulting model explained portions of interindividual variability in melphalan exposure, therapeutic effect, and feedback regulation of G-CSF on neutrophils, thus enabling simulation of various doses and prediction of neutropenia duration.

Visual predictive check (VPC) plot of the final model simulated data vs. observed data in (a) all patients, (b) with granulocyte‐colony stimulating factor (G‐CSF) regimen starting on day +1, and (c) with G‐CSF regimen starting on day +7 after transplantation. Blue dots, the observed data; black dashed line, 2.5th and 97.5th percentiles of the observed data; black solid line, the median of the observed data; red solid line, the median of the simulated data; gray area, 95% prediction interval of the simulated data; black dashed straight line, absolute neutrophil count (ANC) = 0.5 K cells/μL.
Figure: Visual predictive check (VPC) plot of the final model simulated data vs. observed data in (a) all patients, (b) with granulocyte‐colony stimulating factor (G‐CSF) regimen starting on day +1, and (c) with G‐CSF regimen starting on day +7 after transplantation. Blue dots, the observed data; black dashed line, 2.5th and 97.5th percentiles of the observed data; black solid line, the median of the observed data; red solid line, the median of the simulated data; gray area, 95% prediction interval of the simulated data; black dashed straight line, absolute neutrophil count (ANC) = 0.5 K cells/μL.

Cho YK, Irby DJ, Li J, Sborov DW, Mould DR, Badawi M, Dauki A, Lamprecht M, Rosko AE, Fernandez S, Hade EM, Hofmeister CC, Poi M, Phelps MA. Pharmacokinetic-Pharmacodynamic Model of Neutropenia in Patients With Myeloma Receiving High-Dose Melphalan for Autologous Stem Cell Transplant. CPT Pharmacometrics Syst Pharmacol. 2018 Nov;7(11):748-758. doi: 10.1002/psp4.12345. Epub 2018 Oct 20. PMID: 30343510; PMCID: PMC6263666.

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Cachectic Cancer Patients: Immune to Checkpoint Inhibitor Therapy?

Immune checkpoint inhibition is dramatically improving patient outcomes in diverse cancers, many of which responded poorly to traditional cytotoxic agents. Drivers of heterogeneous response to immune checkpoint therapy are poorly characterized. Cachectic cancer patients exhibit elevated pembrolizumab clearance and poor response, highlighting the immense therapeutic challenge posed by cancer cachexia. See related article by Turner et al., p. 5841.

Figure 1: Cachexia/malnutrition blocks the beneficial effects of pembrolizumab independent of pembrolizumab exposure.
Figure 1: Cachexia/malnutrition blocks the beneficial effects of pembrolizumab independent of pembrolizumab exposure.

Coss CC, Clinton SK, Phelps MA. Cachectic Cancer Patients: Immune to Checkpoint Inhibitor Therapy? Clin Cancer Res. 2018 Dec 1;24(23):5787-5789. doi: 10.1158/1078-0432.CCR-18-1847. Epub 2018 Jul 17. PMID: 30018117; PMCID: PMC6279566.

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Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells

Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. *p < 0.05.
Figure: Measurements of different cell populations in spleen cells by flow cytometry using appropriate surface markers conjugated with different fluorochromes. Fluorescence signal of T-cell surface marker CD3e conjugated with PE-Vio770 (P1 = T cells) and B-cell surface marker CD19 conjugated with APC-Vio770 (P2 = B cells) on the cells from mice receiving (a) vehicle control or (b) lipopolysaccharide (LPS). The percentage of each cell population in mice (c) 24 h after LPS treatment (three doses, n = 4) or (d) 3 weeks after treatment with extracellular vesicles (10 doses, n = 10). Bars and error bars denote the mean and standard deviation, respectively, of experimental groups. PBS, phosphate-buffered saline; WT, wild-type HEK293T cells. *p < 0.05.

Zhu X, Badawi M, Pomeroy S, Sutaria DS, Xie Z, Baek A, Jiang J, Elgamal OA, Mo X, Perle K, Chalmers J, Schmittgen TD, Phelps MA. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J Extracell Vesicles. 2017 Jun 6;6(1):1324730. doi: 10.1080/20013078.2017.1324730. PMID: 28717420; PMCID: PMC5505007.

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