Janet L. Markman

Nanomedicine therapeutic approaches to overcome cancer drug resistance

Nanomedicine is an emerging form of therapy that focuses on alternative drug delivery and improvement of the treatment efficacy while reducing detrimental side effects to normal tissues. Cancer drug resistance is a complicated process that involves multiple mechanisms. Here we discuss the major forms of drug resistance and the new possibilities that nanomedicines offer to overcome these treatment obstacles. Novel nanomedicines that have a high ability for flexible, fast drug design and production based on tumor genetic profiles can be created making drug selection for personal patient treatment much more intensive and effective. This review aims to demonstrate the advantage of the young medical science field, nanomedicine, for overcoming cancer drug resistance. With the advanced design and alternative mechanisms of drug delivery known for different nanodrugs including liposomes, polymer conjugates, micelles, dendrimers, carbon-based, and metallic nanoparticles, overcoming various forms of multi-drug resistance looks promising and opens new horizons for cancer treatment.

Polymalic acid nanobioconjugate for simultaneous immunostimulation and inhibition of tumor growth in HER2/neu-positive breast cancer.

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody-cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensure the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform.

Nanobiopolymer for Direct Targeting and Inhibition of EGFR Expression in Triple Negative Breast Cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.

Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment

Abstract Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient’s treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-l-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconjugate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.

Gene expression changes in rat brain after short and long exposures to particulate matter in Los Angeles basin air: Comparison with human brain tumors.

Air pollution negatively impacts pulmonary, cardiovascular, and central nervous systems. Although its influence on brain cancer is unclear, toxic pollutants can cause blood-brain barrier disruption, enabling them to reach the brain and cause alterations leading to tumor development. By gene microarray analysis validated by quantitative RT-PCR and immunostaining we examined whether rat (n=104) inhalation exposure to air pollution particulate matter (PM) resulted in brain molecular changes similar to those associated with human brain tumors. Global brain gene expression was analyzed after exposure to PM (coarse, 2.5-10μm; fine, <2.5μm; or ultrafine, <0.15μm) and purified air for different times, short (0.5, 1, and 3 months) and chronic (10 months), for 5h per day, four days per week. Expression of select gene products was also studied in human brain (n=7) and in tumors (n=83). Arc/Arg3.1 and Rac1 genes, and their protein products were selected for further examination. Arc was elevated upon two-week to three-month exposure to coarse PM and declined after 10-month exposure. Rac1 was significantly elevated upon 10-month coarse PM exposure. On human brain tumor sections, Arc was expressed in benign meningiomas and low-grade gliomas but was much lower in high-grade tumors. Conversely, Rac1 was elevated in high-grade vs. low-grade gliomas. Arc is thus associated with early brain changes and low-grade tumors, whereas Rac1 is associated with long-term PM exposure and highly aggressive tumors. In summary, exposure to air PM leads to distinct changes in rodent brain gene expression similar to those observed in human brain tumors.

Resistance to receptor-blocking therapies primes tumors as targets for HER3-homing nanobiologics

ABSTRACT Resistance to anti‐tumor therapeutics is an important clinical problem. Tumor‐targeted therapies currently used in the clinic are derived from antibodies or small molecules that mitigate growth factor activity. These have improved therapeutic efficacy and safety compared to traditional treatment modalities but resistance arises in the majority of clinical cases. Targeting such resistance could improve tumor abatement and patient survival. A growing number of such tumors are characterized by prominent expression of the human epidermal growth factor receptor 3 (HER3) on the cell surface. This study presents a “Trojan‐Horse” approach to combating these tumors by using a receptor‐targeted biocarrier that exploits the HER3 cell surface protein as a portal to sneak therapeutics into tumor cells by mimicking an essential ligand. The biocarrier used here combines several functions within a single fusion protein for mediating targeted cell penetration and non‐covalent self‐assembly with therapeutic cargo, forming HER3‐homing nanobiologics. Importantly, we demonstrate here that these nanobiologics are therapeutically effective in several scenarios of resistance to clinically approved targeted inhibitors of the human EGF receptor family. We also show that such inhibitors heighten efficacy of our nanobiologics on naïve tumors by augmenting HER3 expression. This approach takes advantage of a current clinical problem (i.e. resistance to growth factor inhibition) and uses it to make tumors more susceptible to HER3 nanobiologic treatment. Moreover, we demonstrate a novel approach in addressing drug resistance by taking inhibitors against which resistance arises and re‐introducing these as adjuvants, sensitizing tumors to the HER3 nanobiologics described here. Graphical abstract Figure. Tumor cells with resistance to HER2 inhibitors can be prime targets for HER3‐directed nanobiologics. Schematic summarizes several scenarios that may explain how drug‐resistant tumor cells can be targeted by HER3‐nanobiologics. In the first scenario, tumor cells with acquired resistance to HER2‐targeted inhibitors are characterized by increased cell surface display of HER3, cornering these resistant cells for attack by the HER3‐nanobiologics described here. These studies also suggest that pre‐treatment of sensitive or naïve tumors may yield the same result by shifting naïve tumor cells to a HER3‐elevated phenotype. In the second scenario, HER2‐sensitive tumor cells in a heterogenous population may be eliminated, leaving resistant cells that could be vulnerable to nanobiologic attack due to increased cell surface display of HER3. The studies described here also suggest that such tumors with inherent or pre‐existing resistance to HER2 inhibitors may likely have increased cell surface HER3, thus making them vulnerable to nanobiologic attack.

T Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation

&NA; Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin‐17A‐mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on ROR&agr; transcription factor and interleukin‐1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell‐permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell‐intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses. Graphical Abstract Figure. No caption available. HighlightsRIP2 deficiency in CD4+ T cells leads to severe IL‐17A‐mediated diseasesRIP2 suppresses pathogenic Th17 cell and supports conventional Th17 cell polarizationRIP2 suppression of pathogenic Th17 cell differentiation is ROR&agr; and IL‐1 dependentRIP2 CARD domain regulates pathogenic Th17 cell differentiation in CD4+ T cells &NA; RIP2 is the key adaptor molecule for NOD1‐ and NOD2‐mediated intracellular signaling to sense pathogens and cell activation in myeloid cells. Shimada, Porritt, and colleagues demonstrate a previously unappreciated role for RIP2 in Th17 cell regulation and differentiation in a T cell‐intrinsic manner.

Abstract 1459: Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone

Introduction: At advanced age, men have increasingly higher incidence of melanoma compared with women, as well as continuously decreasing levels of testosterone. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the immunological and hormonal mechanisms underlying the gender disparity are warranted. Procedures: To investigate the gender disparity observed between men and women affected by melanoma, we used an experimental murine model where B16 melanoma cells were injected i.v. into C57BL/6 mice and lung colonization was assessed at day 14. The roles of neutrophils and NK cells were then investigated by specific cell depletion using anti-Ly6G(1A8) and anti-NK1.1 monoclonal antibodies, respectively. Depleting antibodies or control IgG were injected every 3 days, starting one day before melanoma cell injection. To elucidate the role of hormones, ovariectomy or castration surgeries were performed 4 weeks before B16 injection and 60 day slow release pellets were inserted at the time of surgery. Findings were reproduced with a second murine melanoma cell line (YUMM1.7) derived from a genetically engineered mouse harboring human mutations in BrafV600, as well as loss of Cdkn2a and PTEN. Data: Compared with males, naive female mice injected with melanoma (B16 or YUMM1.7) cells have higher lung tumor burden, reduced neutrophil infiltration, and decreased NK cell activation. Ovariectomy of female mice did not affect lung tumor burden, indicating that estrogen and progesterone are not protective. Interestingly, castrated males revealed increased lung metastasis and worse survival rates compared with sham male mice, suggesting a protective role for testosterone. Further, testosterone replacement in castrated mice significantly reduced the elevated lung tumor burden to equal levels observed in sham males. Depletion of NK cells greatly increased tumor burden in both male and female mice in a gender independent manner. In contrast, neutrophil depletion increased lung tumor burden only in male mice and had no significant affect in female mice, indicating a potential gender difference in neutrophils. Moreover, neutrophil depletion in male mice also reduced NK activation and IFN-γ production, suggesting a stimulatory relationship between neutrophils and NK cells. Conclusion: Our data indicates that both neutrophils and NK cells are important for the initial response against melanoma colonization of the lung. Neutrophil depletion, as well as our castration data, indicate that there is a gender specific differential response, which may contribute to the gender disparity seen in human melanoma incidence and survival. Further, testosterone replacement at physiological levels may benefit a subset of melanoma patients. Citation Format: Janet Markman, Daiko Wakita, Timothy Crother, Moshe Arditi. Revealing the underlying causes of the gender disparity in melanoma: Role of testosterone. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1459.

Abstract 4057: Gender and the immune system in lung and liver colonization of murine melanoma in a B16 metastatic model

Introduction: Until age 45, incidence rates of melanoma are higher in woman than men, but by age 60, rates in men are more than double and by age 80, they are almost triple. With the low rate of survival for distant metastatic patients with melanoma, especially in men, studies aimed at elucidating the mechanisms underlying metastasis and the gender disparity are warranted. The purpose of this study is to identify the immune cells and cellular interactions that are crucial for initial colonization of metastatic melanoma in a gender dependent manner. Experimental Procedures: We used a B16 melanoma metastatic model in which 2×105 cells are injected via the tail vein of B6 mice and allowed to colonize for 14 days. Positive organs are analyzed by visual counting of the metastases, extraction of the melanin pigment and measurement by spectrophotometry, and by flow cytometry. To investigate the role of innate immunity, we injected ip either anti-Ly6G, 1A8 (neutrophil depletion), anti NK1.1, PK136 (natural killer [NK] cell depletion) or control IgG every 3 days, starting 1 day before tumor injection. For hormone ablation studies, an LHRH antagonist was injected. Data: Naive mice injected with B16 cells revealed that females have higher lung tumor burden than males. Previous castration studies indicate that this may be dependent on androgens. Intriguingly, neutrophil depletion increases tumor burden only in male mice when compared to male IgG and does not significantly affect tumor burden in female mice, indicating a potential gender difference in neutrophils. Neutrophil depletion in male mice also reduced NK activation and IFN-γ production, indicating a stimulatory relationship between neutrophils and NK cells. A subsequent NK depletion study revealed significantly increased tumor burden in both male and female NK-depleted mice when compared to the gender-matched controls. However, the increased tumor burden normally seen in lungs of females compared to males was still apparent when comparing lungs of NK depleted male mice to NK depleted female mice. This study also revealed that NK cells are crucial for the prevention of liver metastasis as both genders of NK depleted mice revealed multiple melanoma tumor nodules on the surface of the liver. Conclusion: Our data indicates that both neutrophils, and to a greater extent, NK cells are important for the initial response against B16 melanoma colonization of the lung and may be necessary to prevent metastasis. For the liver, NK cells were absolutely crucial for the prevention of melanoma colonization in this organ. Neutrophil depletion, as well as our previous castration data, indicated that there is a gender specific differential response of these cells, which may contribute to the gender disparity seen in human incidence and survival. Further, the importance of NK cells in killing of tumor cells indicates a population of patients that may benefit from ex vivo NK expansion therapy currently in clinical trials. Citation Format: Janet L. Markman, Daiko Wakita, Timothy R. Crother, Moshe Arditi. Gender and the immune system in lung and liver colonization of murine melanoma in a B16 metastatic model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4057. doi:10.1158/1538-7445.AM2015-4057

Abstract A50: Nanobiocojugates of differential imaging and treatment of brain metastatic tumors

Introduction: A significant clinical problem with brain metastatic (BM) tumors is drug delivery and diagnostic imaging to verify MRI enhancement(s) for planning treatment. MRI enhancement in cancer patient9s brain might result from infection after chemotherapy that impairs immune system; metastasis from primary lung/breast cancer; or a new primary brain tumor. Unlike lung/breast, brain biopsies are often technically impossible. Therefore, there is urgent need for the development of effective theranostic (dual therapy and diagnostic) systems against brain metastatic cancer. Most chemotherapeutic drugs or therapeutic monoclonal antibodies (mAb), Trastuzumab, Cetuximab, and Rituximab, are effective for primary tumor treatment but cannot penetrate blood brain barrier (BBB) failing to treat brain metastasis. We used a natural nanobiopolymer, polymalic acid (PMLA), as a nanoplatform for the family of tumor-targeted PolycefinTM drugs to provide differential brain tumor imaging and treatment. Methods: Three xenogeneic orthotropic human brain metastatic tumors, MDA-MB-474, HER2+ breast cancer; A549 lung cancer, and MDA-MB-468, triple negative breast cancer (TNBC), both EGFR+, were inoculated stereotactically into the brain of mice. For diagnostic imaging, PolycefinTM was used with covalently attached MRI tracer Gadolinium (Gd-DOTA). Morpholino antisense oligonucleotides (AON) were conjugated to PolycefinTM to specifically inhibit gene/protein expression to block tumor growth. The combination of cell surface targeting mAbs, including anti-transferrin receptor (TfR) mAb for drug BBB transcytosis, and AONs to multiple tumor markers on the same delivery polymer was used for anti-tumor treatment. MRI 1H imaging was performed on a 9.4-Tesla small animal MRI system. Treatment groups of animals included (1) HER2+ MDA-MB-474 breast cancer metastases targeted with PMLA-Gd-DOTA/HER2 mAb/TfR mAb; (2) EGFR+ MDA-MB-468 TNBC metastases targeted with PMLA-Gd-DOTA/EGFR mAb/TfR mAb; and (3) Controls for all treatments inoculated with PMLA-IgG mAb and clinical Gd. Unpublished Results. Imaging: Dynamic T1 analysis. Similar data for specific tumor imaging were obtained for brain-implanted lung and breast tumors: the inverse of T1-1 relaxation time (proportional to Gd concentration) was measured in healthy brain part and in the tumor. T1-1 time dependence for Gd-DOTA-Polycefin (T1-1 ratio tumor/normal brain) was compared with clinically used Gd MRI agent, MultiHance®. After reaching a maximum, high T1-1 relative values prevailed for several hours for Gd-DOTA-mAb-Polycefin, but declined rapidly for Gd. High contrast for Gd was seen in 20 min, whereas that for Gd-DOTA-Polycefin peaked in 45-60 min, and remained for up to 3 hrs. By differential MRI with anti-HER2 (Trastuzumab) or anti-EFGR (Cetuximab) mAb attached covalently to the nanoplatform, we were able to differentiate HER2+ from EGFR+ metastatic brain tumors with corresponding imaging controls. Treatment: Animal survival after Polycefin treatment of various brain metastases was significantly higher than in untreated (PBS) or therapeutic mAb (Herceptin or Cetuximab) treated animals. These survival increases were as follows: 66% for lung cancer metastasis, 47% for HER2+ breast cancer metastasis, and 81% for TNBC metastasis. Conclusions. We have developed a system for differential imaging and treatment of various metastatic brain tumors based on specific metastasis targeting, and inhibition of expression of tumor-specific genes/proteins. Systemic treatment with this system resulted in significantly increased survival of brain metastatic tumor-bearing animals. Citation Format: Julia Y. Ljubimova, R. Patil, P. Gangalum, S. Wagner, S. Inoue, H. Ding, J. Portilla, K. Rekechenetskiy, K. Bindu, J. Markman, A. Chesnokova, K. L. Black, E. Holler. Nanobiocojugates of differential imaging and treatment of brain metastatic tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A50.