Hydrogel-mediated delivery of celastrol and doxorubicin induces a synergistic effect on tumor regression via upregulation of ceramides.

PDXs recapitulate histologic features, gene expression patterns, and genomic alterations in human primary tumors, and thus have emerged as robust preclinical models for drug development, molecular characterization of cancers, identification of biomarkers, and strategic development of precision therapy. We tested multiple combinations of small molecule targeted drugs selected on the basis of known NSCLC vulnerabilities for efficacy in NSCLC PDXs with known genotypes. We determined in vivo treatment responses to single agent and combination therapies for pathway targeted therapeutic agents, including the MEK inhibitor trametinib, the MDM2 inhibitor KRT-232, the BCL2/BCL-XL inhibitor navitoclax, and their combinations in 8-23 molecularly annotated NSCLC PDX models. Mice (n=3-5/group) were enrolled into treatment individually when tumors reached 200 mm3 in size, and were treated 5 days/week for 3 weeks. Tumor growth was monitored 2-3 times/week. We used the following criteria to determine treatment responses: 1) Tumor Regression (or partial response): tumor regression ≥ -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline (beginning of treatment at day 0); 2) Tumor Growth Inhibition (or stable disease): Tumor growth was significantly suppressed when compared with control (P < 0.05), but no tumor regression was observed, or tumor regression was less than -30% based on tumor volume changes calculated by AUC0-21day or at day 21 after treatment start when compared with baseline; 3) Resistance: Tumor volume changes calculated by AUC0-21day was not significantly different from control group (P>0.05). Our results showed that KRT-232 alone resulted in 15.8% (3/19) tumor regression and 26.3% (5/19) tumor growth inhibition, all in TP53 wild type PDXs. Trametinib alone induced 10% (2/20) tumor regression and 50% (10/20) growth inhibition, respectively. 80% (8/10) of KRAS mutant PDXs responded to trametinib treatment with tumor regression (1/10) or growth inhibition (7/10). Combination therapies of trametinib plus KRT-232 and trametinib plus navitoclax led to improved in vivo anticancer activity over single agent activity in a subset PDX models with KRAS mutations. Tumor regression was observed in 26% (6/23) and 50% (5/10) of trametinib plus KRT-232 and trametinib plus navitoclax treatment groups, respectively. Navitoclax alone did not induce tumor regression in 8 PDX models tested, and navitoclax plus KRT-232 did not lead to significant improvement in activity over single agents in 11 PDXs. Our results show that combination therapies of trametinib plus KRT-232 or navitoclax result in improved efficacy in a subgroup of NSCLC PDX models with KRAS mutations. Clinical trials with these targeted drug combinations in NSCLC are warranted. Citation Format: Xiaoshan Zhang, Ran Zhang, Huiqin Chen, Li Wang, Chenghui Ren, Shuhong Wu, Min Jin Ha, Jeffrey Morris, Yuanxin Xi, Jing Wang, Don L. Gibbons, John V. Heymach, Funda Meric-Bernstam, John Minna, Stephen G. Swisher, Jack A. Roth, Bingliang Fang. Novel targeted combination therapies active in KRAS mutant non-small cell lung cancer (NSCLC) identified using patient-derived xenografts (PDX) [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 557.

Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. Mutations of the PIK3CA gene, which encodes the class I PI3K catalytic subunit p110, are found in many cancer patients and activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel inhibitor, selective to class I PI3K and with potent antitumor activity against tumors harboring PIK3CA mutations. Here we investigated the relationship between in vivo efficacy and PI3K signaling inhibition. CH5132799 suppressed tumor growth in a wide variety of xenograft models with PI3K pathway activation, including those with PIK3CA mutations. CH5132799 has two modes of tumor growth inhibition, tumor regression and tumor stasis. On the one hand, in the breast cancer xenograft models harboring PIK3CA mutations (KPL-4 and BT-474), CH5132799 induced remarkable tumor regression and also shrank the tumor of PTEN-deleted prostate cancer GXF-97 (‘tumor regression models’). On the other hand, in colorectal cancers with both PIK3CA and KRAS mutations (HCT116 and HCT15), CH5132799 treatment resulted in tumor stasis but did not induce tumor regression (‘tumor stasis models’). We analyzed PI3K and MAPK signaling in these xenograft tumors to explore the factors which could explain the tumor growth inhibition modes of CH5132799, tumor regression or stasis. Phosphorylation of Akt and S6 was inhibited by CH5132799 administration in all the tested tumors but ERK phosphorylation was not. Notably, 4E-BP1 phosphorylation (p-4E-BP1) was well suppressed in the tumor regression models but was only partially decreased in the tumor stasis models. These findings suggest that p-4E-BP1 determines whether CH5132799 induces tumor regression or stasis. Since p-4E-BP1 is known to be regulated by both PI3K and MAPK pathways, we examined the effects on p-4E-BP1 of combining inhibitors selective to each pathway: CH5132799 and RO4987655, an allosteric MEK inhibitor. Compared to their effect as single agents, the combined inhibitors drastically suppressed p-4E-BP1 in HCT116 cells in vitro, leading to potent growth inhibition and apoptosis induction. Furthermore, combining CH5132799 and RO4987655 induced remarkable in vivo tumor regression even in the HCT116 tumor stasis model, confirming the significance of p-4E-BP1 suppression in CH5132799-induced tumor regression. In conclusion, tumor regression or stasis induced by CH5132799 correlated with p-4E-BP1 being suppressed in the tumors. Moreover, in the model in which CH5132799 alone had induced tumor stasis with partial p-4E-BP1 suppression, combination with the MEK inhibitor achieved p-4E-BP1 suppression and tumor regression. From these lines of preclinical evidence, p-4E-BP1 suppression could also be a determinant factor of the CH5132799 response in clinical settings, and combination therapy with MEK inhibitors is a promising strategy to enhance CH5132799 efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B157.

Th1 adjuvant ARNAX, in combination with radiation therapy, enhances tumor regression in mouse tumor-implant models

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

Redox responsive biodegradable polymersomes comprising of poly(ethylene glycol)-polylactic acid-poly(ethylene glycol) [PEG-s-s-PLA-s-s-PLA-s-s-PEG] triblock copolymer with multiple disulfide linkages were developed to improve intracellular delivery and to enhance chemotherapeutic efficacy of doxorubicin in breast cancer with minimal cardiotoxicity. Folic acid and trastuzumab functionalized monodispersed polymersomes of size ∼150 nm were prepared by nanoprecipitation method while achieving enhanced doxorubicin loading of ∼32% in the polymersomes. Multiple redox responsive disulfide linkages were incorporated in the polymer in order to achieve complete disintegration of polymersomes in redox rich environment of cancer cells resulting in enhanced doxorubicin release as observed in in vitro release studies, where ∼90% doxorubicin release was achieved in pH 5.0 in the presence of 10 mM glutathione (GSH) as compared to ∼20% drug release in pH 7.4. Folic acid and trastuzumab mediated active targeting resulted in improved cellular uptake and enhanced apoptosis in in vitro studies in breast cancer cell lines. In vivo studies in Ehrlich ascites tumor bearing Swiss albino mice showed enhanced antitumor efficacy and minimal cardiotoxicity of polymersomes with ∼90% tumor regression as compared to ∼38% tumor regression observed with free doxorubicin. The results highlight therapeutic potential of the polymersomes as doxorubicin delivery nanocarrier in breast cancer therapy with its superior antitumor efficacy and minimal cardiotoxicity.

To evaluate the influence of subtotal radiofrequency (RF) ablation on a tumor-specific immune response in a murine tumor model and to explore the role of intratumoral dendritic cells (ITDCs) in mediating this effect. Animal work was performed according to an approved protocol and in compliance with the National Cancer Institute Animal Care and Use Committee guidelines and regulations. A murine urothelial carcinoma (MB49) model expressing the male minor histocompatibility (HY) antigen was inoculated subcutaneously in female mice. Fourteen days later, splenic T cells were analyzed with enzyme-linked immunosorbent spot for HY immune response (n = 57). In subsequent experiments, mice were randomized into control (n = 7), RF ablation, ITDC (n = 9), and RF ablation + ITDC (n = 9) groups and monitored for tumor growth. Eleven days after treatment, tumors were harvested for histologic and immunohistochemical analysis. Animals demonstrating complete tumor regression were rechallenged in the contralateral flank. Animals treated with subtotal RF ablation showed significant increases in tumor-specific class I and II responses to HY antigens and tumor regression. RF ablation, ITDC, and combined groups demonstrated similar levels of antigen-presenting cell infiltration; all groups demonstrated greater levels of infiltration compared with untreated controls. ITDC injection also resulted in tumor regression. However, combination therapy did not enhance tumor regression when compared with either treatment alone. Rechallenged mice in RF ablation, ITDC, and combination groups demonstrated significant tumor growth inhibition compared with controls. Subtotal RF ablation treatment results in enhanced systemic antitumor T-cell immune responses and tumor regression that is associated with increased dendritic cell infiltration. ITDC injection mimics the RF ablation effect but does not increase immune responses when injected immediately after RF ablation.

To determine if pulsed-high intensity focused ultrasound (HIFU) could effectively serve as a source of hyperthermia with thermosensitive liposomes to enhance delivery and efficacy of doxorubicin in tumors. Comparisons in vitro and in vivo were carried out between non-thermosensitive liposomes (NTSL) and low temperature-sensitive liposomes (LTSL). Liposomes were incubated in vitro over a range of temperatures and durations, and the amount of doxorubicin released was measured. For in vivo experiments, liposomes and free doxorubicin were injected i.v. in mice followed by pulsed-HIFU exposures in s.c. murine adenocarcinoma tumors at 0 and 24 h after administration. Combinations of the exposures and drug formulations were evaluated for doxorubicin concentration and growth inhibition in the tumors. In vitro incubations simulating the pulsed-HIFU thermal dose (42 degrees C for 2 min) triggered release of 50% of doxorubicin from the LTSLs; however, no detectable release from the NTSLs was observed. Similarly, in vivo experiments showed that pulsed-HIFU exposures combined with the LTSLs resulted in more rapid delivery of doxorubicin as well as significantly higher i.t. concentration when compared with LTSLs alone or NTSLs, with or without exposures. Combining the exposures with the LTSLs also significantly reduced tumor growth compared with all other groups. Combining low-temperature heat-sensitive liposomes with noninvasive and nondestructive pulsed-HIFU exposures enhanced the delivery of doxorubicin and, consequently, its antitumor effects. This combination therapy could potentially produce viable clinical strategies for improved targeting and delivery of drugs for treatment of cancer and other diseases.

<div>Abstract<p><b>Purpose:</b> In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC).</p><p><b>Experimental Design:</b> Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. PSA measurements obtained before, and during, therapy were used. Data analysis using a two-phase mathematical equation yielded concomitant PSA growth and regression rate constants.</p><p><b>Results:</b> Growth rate constants (<i>g</i>) can be estimated while patients receive therapy and in such patients <i>g</i> is superior to PSA-DT in predicting OS. Incremental reductions in growth rate constants were recorded in successive trials with a 10-fold slower <i>g</i> in the most recent combination therapy trial (log <i>g</i> = 10<sup>−3.17</sup>) relative to single-agent thalidomide (log <i>g</i> = 10<sup>−2.08</sup>) more than a decade earlier. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration.</p><p><b>Conclusion:</b> Incremental reductions in tumor growth rate constants suggest increased efficacy in successive chemotherapy trials. The derived growth rate constant correlates with survival, and may be used to assess efficacy. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. If validated as a surrogate for survival, growth rate constants would offer an important new efficacy endpoint for clinical trials. <i>Clin Cancer Res; 17(4); 907–17. ©2010 AACR</i>.</p></div>

In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC). Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. PSA measurements obtained before, and during, therapy were used. Data analysis using a two-phase mathematical equation yielded concomitant PSA growth and regression rate constants. Growth rate constants (g) can be estimated while patients receive therapy and in such patients g is superior to PSA-DT in predicting OS. Incremental reductions in growth rate constants were recorded in successive trials with a 10-fold slower g in the most recent combination therapy trial (log g = 10(-3.17)) relative to single-agent thalidomide (log g = 10(-2.08)) more than a decade earlier. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration. Incremental reductions in tumor growth rate constants suggest increased efficacy in successive chemotherapy trials. The derived growth rate constant correlates with survival, and may be used to assess efficacy. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. If validated as a surrogate for survival, growth rate constants would offer an important new efficacy endpoint for clinical trials.

The tumour vasculature is essential for maintaining tumour growth and regulating the tumour microenvironment through the supply of nutrients and oxygen. ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid, DMXAA) is a flavonoid, non tubulin-binding Tumour-Vascular Disrupting Agent (Tumour-VDA) that induces breakdown of the established tumour vasculature resulting in the inhibition of tumour blood supply leading to tumour ischemia and extensive necrosis of the tumour core. Unlike anti-angiogenics, ASA404 has limited effects on angiogenesis at the tumour periphery and as a consequence the remaining peripheral tumour rim represents a source for regrowth of the tumour upon cessation of ASA404 treatment. Preclinical and clinical studies have highlighted the effectiveness of combining chemotherapies, especially taxanes, with ASA404 for marked tumour inhibition. In the present studies, the influence of adding ASA404 to taxane-containing regimens of trastuzumab and bevacizumab was examined in breast cancer xenograft models. The effects of the combination of trastuzumab plus paclitaxel +/- ASA404 was examined using the BT-474 breast cancer model in C.B-17 SCID mice. Adminstration schedules were as follows: ASA404 (15 and 20 mg/kg iv, once daily on days 1, 5 and 9), paclitaxel (P) (10 and 15 mg/kg iv, qod x5) and trastuzumab (T) (10 mg/kg ip, biwk x4). Efficacy was assessed on day 27 as the % T/C (growth inhibition) or % mean reduction from the mean tumour volume at day 1 (T/To; regression of tumour volume). ASA404 alone showed no anti-tumour effects. Dual combination of P+T at 15 and 10 mg/kg respectively produced significant tumour inhibition with 21% T/C. Combination of ASA404 at 15 or 20 mg/kg with P (15 mg/kg) and T (10 mg/kg) resulted in significant tumour inhibition (11% T/C) and regression (45% T/To) being observed respectively. The 20 mg/kg ASA404-containing triple combination therapy significantly improved the efficacy compared to the corresponding ASA404 monotherapy or P+T dual therapy and yielded partial regressions in 3 of 11 mice by day 27. The effects of the combination of bevacizumab plus docetaxel +/- ASA404 was examined using the MCF-7 breast cancer model in nu/nu nude mice. Adminstration schedules were as follows: ASA404 (20 mg/kg iv, once daily on days 1, 5 and 9), docetaxel (D) (4 and 6 mg/kg iv, qwk x3) and bevacizumab (B) (10 mg/kg ip, biwk x4). Efficacy was assessed on day 17. ASA404 alone had a modest, non-significant anti-tumour effect (64% T/C). Dual combination of B with D at 4 or 6 mg/kg resulted in 28% and 13 % T/C respectively. The triple combination of ASA404 with B+D (4 or 6 mg/kg) resulted in tumour regressions (8% and 5% T/To respectively), although this observed improvement of the triple therapy over the B+D combination or ASA404 alone was not statistically signifcant. These data indicate the potential value for the combination of ASA404 (vadimezan) to the taxane-containing regimens of trastuzumab and bevacizumab in these preclinical breast cancer models. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3142.

<div>Abstract<p><b>Purpose:</b> In solid tumors such as prostate cancer, novel paradigms are needed to assess therapeutic efficacy. We utilized a method estimating tumor growth and regression rate constants from serial PSA measurements, and assessed its potential in patients with metastatic castration resistant prostate carcinoma (mCRPC).</p><p><b>Experimental Design:</b> Patients were enrolled in five phase II studies, including an experimental vaccine trial, representing the evolution of therapy in mCRPC. PSA measurements obtained before, and during, therapy were used. Data analysis using a two-phase mathematical equation yielded concomitant PSA growth and regression rate constants.</p><p><b>Results:</b> Growth rate constants (<i>g</i>) can be estimated while patients receive therapy and in such patients <i>g</i> is superior to PSA-DT in predicting OS. Incremental reductions in growth rate constants were recorded in successive trials with a 10-fold slower <i>g</i> in the most recent combination therapy trial (log <i>g</i> = 10<sup>−3.17</sup>) relative to single-agent thalidomide (log <i>g</i> = 10<sup>−2.08</sup>) more than a decade earlier. Growth rate constants correlated with survival, except in patients receiving vaccine-based therapy where the evidence demonstrates prolonged survival presumably due to immunity developing subsequent to vaccine administration.</p><p><b>Conclusion:</b> Incremental reductions in tumor growth rate constants suggest increased efficacy in successive chemotherapy trials. The derived growth rate constant correlates with survival, and may be used to assess efficacy. The PSA-TRICOM vaccine appears to have provided marked benefit not apparent during vaccination, but consistent with subsequent development of a beneficial immune response. If validated as a surrogate for survival, growth rate constants would offer an important new efficacy endpoint for clinical trials. <i>Clin Cancer Res; 17(4); 907–17. ©2010 AACR</i>.</p></div>

BackgroundMost current cancer immunotherapies are based on mobilizing CD8 T cell responses. However, many types of tumors evade CD8 T cell recognition by displaying few or no antigens, or losing expression of MHC I. These considerations underlie the need for complementary therapies that mobilize other antitumor effector cells, such as NK cells, which preferentially kill MHC I-deficient cells. Cyclic dinucleotides (CDNs) activate the cGAS-STING pathway of the innate immune system and are candidates as immunotherapy agents. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce tumor regression [1]. CDN therapy also induces long-term tumor regressions in some MHC I-deficient tumor models, mediated primarily by NK cells [2].MethodsTo extend the efficacy of CDN therapy, we combined the IL-2 superkine, H9, or half-life extended H9, with CDNs to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of NK cell desensitization [3,4]. In these studies, we utilized B16-F10 and MC38 tumor cells lacking B2m to examine effects of the combination therapy on MHC I-deficient tumor growth as well as to examine the activation of NK cells by flow cytometry and cytotoxicity assays. We also utilized B16-F10 WT and the spontaneous tumor model, MCA, to assess the effect of the combination therapy on MHC I+ tumors.ResultsHere we show that H9 synergized with CDN therapy to mobilize much more powerful antitumor responses against MHC I-deficient tumors than CDN alone. The responses were mediated by NK cells and in some cases CD4 T cells, and were accompanied by increased recruitment to and sustained activation of NK cells in the tumor. This combination therapy regimen activated NK cells systemically, as shown by antitumor effects distant from the site of CDN injection and enhanced cytolytic activity of splenic NK cells against tumor cell targets ex vivo. Finally, the same combination therapy regimen synergistically mobilized powerful CD8 T cell responses in the case of MHC I+ tumor cells, suggesting the generality of the approach. The approach was effective against primary sarcomas, as well, especially when combined with checkpoint therapy, leading to tumor regressions and long-term survival of many mice with MCA-induced sarcoma.ConclusionsOverall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK and T cell-mediated antitumor activity, providing important justification for evaluating this approach for treating cancers that are refractory to available treatment options.ReferencesCorrales, L., Glickman, L.H., McWhirter, S.M., Kanne, D.B., Sivick, K.E., Katibah, G.E., Woo, S.R., Lemmens, E., Banda, T., Leong, J.J., et al. (2015). Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity. Cell Rep 11, 1018–1030.Nicolai, C.J., Wolf, N., Chang, I.C., Kirn, G., Marcus, A., Ndubaku, C.O., McWhirter, S.M., and Raulet, D.H. (2020). NK cells mediate clearance of CD8(+) T cell-resistant tumors in response to STING agonists. Science immunology 5, eaaz2738.Levin, A.M., Bates, D.L., Ring, A.M., Krieg, C., Lin, J.T., Su, L., Moraga, I., Raeber, M.E., Bowman, G.R., Novick, P., et al. (2012). Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529–533.Ardolino, M., Azimi, C.S., Iannello, A., Trevino, T.N., Horan, L., Zhang, L., Deng, W., Ring, A.M., Fischer, S., Garcia, K.C., and Raulet, D.H. (2014). Cytokine therapy reverses NK cell anergy in MHC-deficient tumors. J Clin Invest 124, 4781–4794.

Noninvasive Imaging and Radiovirotherapy of Prostate Cancer Using an Oncolytic Measles Virus Expressing the Sodium Iodide Symporter

Can Antitumor Immunity Help to Explain “Oncogene Addiction”?