FLT1 activation in cancer cells promotes PARP-inhibitor resistance in breast cancer.

Acquired resistance to PARP inhibitors (PARPi) remains a treatment challenge for BRCA1/2-mutant breast cancer that drastically shortens patient survival. Although several resistance mechanisms have been identified, none have been successfully targeted in the clinic. Using new PARPi-resistance models of Brca1- and Bard1-mutant breast cancer generated in-vivo, we identified FLT1 (VEGFR1) as a driver of resistance. Unlike the known role of VEGF signaling in angiogenesis, we demonstrate a novel, non-canonical role for FLT1 signaling that protects cancer cells from PARPi in-vivo through a combination of cell-intrinsic and cell-extrinsic pathways. We demonstrate that FLT1 blockade suppresses AKT activation, increases tumor infiltration of CD8+ T cells, and causes dramatic regression of PARPi-resistant breast tumors in a T-cell-dependent manner. Moreover, PARPi-resistant tumor cells can be readily re-sensitized to PARPi by targeting Flt1 either genetically (Flt1-suppression) or pharmacologically (axitinib). Importantly, a retrospective series of breast cancer patients treated with PARPi demonstrated shorter progression-free survival in cases with FLT1 activation at pre-treatment. Our study therefore identifies FLT1 as a potential therapeutic target in PARPi-resistant, BRCA1/2-mutant breast cancer.

Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Probiotic-guided CAR-T cells for solid tumor targeting.

A major challenge facing tumor-antigen targeting therapies such as chimeric antigen receptor (CAR)-T cells is the identification of suitable targets that are specifically and uniformly expressed on heterogeneous solid tumors. By contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be engineered as antigen-independent platforms for therapeutic delivery. To bridge these approaches, we developed a platform of probiotic-guided CAR-T cells (ProCARs), in which tumor-colonizing probiotics release synthetic targets that label tumor tissue for CAR-mediated lysis in situ. This system demonstrated CAR-T cell activation and antigen-agnostic cell lysis that was safe and effective in multiple xenograft and syngeneic models of human and mouse cancers. We further engineered multifunctional probiotics that co-release chemokines to enhance CAR-T cell recruitment and therapeutic response.

Chemokines expressed by engineered bacteria recruit and orchestrate antitumor immunity.

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16K42A) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8+ T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16K42A-induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Design of combination therapy for engineered bacterial therapeutics in non-small cell lung cancer.

Synthetic biology enables the engineering of bacteria to safely deliver potent payloads to tumors for effective anti-cancer therapies. However, a central challenge for translation is determining ideal bacterial therapy candidates for specific cancers and integrating them with other drug treatment strategies to maximize efficacy. To address this, we designed a screening and evaluation pipeline for characterization of bacterial therapies in lung cancer models. We screened 10 engineered bacterial toxins across 6 non-small cell lung cancer patient-derived cell lines and identified theta toxin as a promising therapeutic candidate. Using a bacteria-spheroid co-culture system (BSCC), analysis of differentially expressed transcripts and gene set enrichment revealed significant changes in at least 10 signaling pathways with bacteria-producing theta toxin. We assessed combinatorial treatment of small molecule pharmaceutical inhibitors targeting 5 signaling molecules and of 2 chemotherapy drugs along with bacterially-produced theta toxin and showed improved dose-dependent response. This combination strategy was further tested and confirmed, with AKT signaling as an example, in a mouse model of lung cancer. In summary, we developed a pipeline to rapidly characterize bacterial therapies and integrate them with current targeted therapies for lung cancer.

Enhancing the tropism of bacteria via genetically programmed biosensors.

Engineered bacteria for therapeutic applications would benefit from control mechanisms that confine the growth of the bacteria within specific tissues or regions in the body. Here we show that the tropism of engineered bacteria can be enhanced by coupling bacterial growth with genetic circuits that sense oxygen, pH or lactate through the control of the expression of essential genes. Bacteria that were engineered with pH or oxygen sensors showed preferential growth in physiologically relevant acidic or oxygen conditions, and reduced growth outside the permissive environments when orally delivered to mice. In syngeneic mice bearing subcutaneous tumours, bacteria engineered with both hypoxia and lactate biosensors coupled through an AND gate showed increased tumour specificity. The multiplexing of genetic circuits may be more broadly applicable for enhancing the localization of bacteria to specified niches.

Phase I trial of the TNF-α inhibitor certolizumab plus chemotherapy in stage IV lung adenocarcinomas.

We previously identified a chemotherapy-induced paracrine inflammatory loop that paradoxically mitigates the anti-tumor effect of chemotherapy and triggers metastatic propagation in breast and lung cancer models. Therefore, we sought to further validate and translate these findings into patient care by coupling the anti-TNF-α drug certolizumab pegol with standard cisplatin doublet chemotherapy. Here we first validate the anti-metastatic effect of certolizumab in a liver-metastatic Lewis Lung Carcinoma model. We then evaluate the safety, efficacy, and pharmacodynamic effects of certolizumab with cisplatin and pemetrexed in an open label Phase 1 clinical trial (NCT02120807) of eighteen adult patients with stage IV lung adenocarcinomas. The primary outcome is maximum tolerated dose. Secondary outcomes are response rate and progression-free survival (PFS); pharmacodynamic changes in blood and tumor are evaluated as a correlative outcome. There were nine partial responses among 16 patients evaluable (56%, 95% CI 30 to 80%). The median duration of response was 9.0 months (range 5.9 to 42.6 months) and median PFS was 7.1 months (95% CI 6.3 to NR). The standard 400 mg dose of certolizumab, added to cisplatin and pemetrexed, is well-tolerated and, as a correlative endpoint, demonstrates potent pharmacodynamic inhibition of peripheral cytokines associated with the paracrine inflammatory loop.

A programmable encapsulation system improves delivery of therapeutic bacteria in mice.

Living bacteria therapies have been proposed as an alternative approach to treating a broad array of cancers. In this study, we developed a genetically encoded microbial encapsulation system with tunable and dynamic expression of surface capsular polysaccharides that enhances systemic delivery. Based on a small RNA screen of capsular biosynthesis pathways, we constructed inducible synthetic gene circuits that regulate bacterial encapsulation in Escherichia coli Nissle 1917. These bacteria are capable of temporarily evading immune attack, whereas subsequent loss of encapsulation results in effective clearance in vivo. This dynamic delivery strategy enabled a ten-fold increase in maximum tolerated dose of bacteria and improved anti-tumor efficacy in murine models of cancer. Furthermore, in situ encapsulation increased the fraction of microbial translocation among mouse tumors, leading to efficacy in distal tumors. The programmable encapsulation system promises to enhance the therapeutic utility of living engineered bacteria for cancer.

Targeting S100A9-ALDH1A1-Retinoic Acid Signaling to Suppress Brain Relapse in EGFR-Mutant Lung Cancer.

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) osimertinib has significantly prolonged progression-free survival (PFS) in patients with EGFR-mutant lung cancer, including those with brain metastases. However, despite striking initial responses, osimertinib-treated patients eventually develop lethal metastatic relapse, often to the brain. Although osimertinib-refractory brain relapse is a major clinical challenge, its underlying mechanisms remain poorly understood. Using metastatic models of EGFR-mutant lung cancer, we show that cancer cells expressing high intracellular S100A9 escape osimertinib and initiate brain relapses. Mechanistically, S100A9 upregulates ALDH1A1 expression and activates the retinoic acid (RA) signaling pathway in osimertinib-refractory cancer cells. We demonstrate that the genetic repression of S100A9, ALDH1A1, or RA receptors (RAR) in cancer cells, or treatment with a pan-RAR antagonist, dramatically reduces brain metastasis. Importantly, S100A9 expression in cancer cells correlates with poor PFS in osimertinib-treated patients. Our study, therefore, identifies a novel, therapeutically targetable S100A9-ALDH1A1-RA axis that drives brain relapse. SIGNIFICANCE: Treatment with the EGFR TKI osimertinib prolongs the survival of patients with EGFR-mutant lung cancer; however, patients develop metastatic relapses, often to the brain. We identified a novel intracellular S100A9-ALDH1A1-RA signaling pathway that drives lethal brain relapse and can be targeted by pan-RAR antagonists to prevent cancer progression and prolong patient survival. This article is highlighted in the In This Issue feature, p. 873.

Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies.

Checkpoint inhibitors have revolutionized cancer therapy but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Because of their preferential colonization of tumors, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we engineer a probiotic bacteria system for the controlled production and intratumoral release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) using a stabilized lysing release mechanism. We used computational modeling coupled with experimental validation of lysis circuit dynamics to determine the optimal genetic circuit parameters for maximal therapeutic efficacy. A single injection of this engineered system demonstrated an enhanced therapeutic response compared to analogous clinically relevant antibodies, resulting in tumor regression in syngeneic mouse models. Supporting the potentiation of a systemic immune response, we observed a relative increase in activated T cells, an abscopal effect, and corresponding increases in systemic T cell memory populations in mice treated with probiotically delivered checkpoint inhibitors. Last, we leveraged the modularity of our platform to achieve enhanced therapeutic efficacy in a poorly immunogenic syngeneic mouse model through effective combinations with a probiotically produced cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Together, these results demonstrate that our engineered probiotic system bridges synthetic biology and immunology to improve upon checkpoint blockade delivery.

Aberrant Zip14 expression in muscle is associated with cachexia in a Bard1-deficient mouse model of breast cancer metastasis.

Nearly 80% of advanced cancer patients are afflicted with cachexia, a debilitating syndrome characterized by extensive loss of muscle mass and function. Cachectic cancer patients have a reduced tolerance to antineoplastic therapies and often succumb to premature death from the wasting of respiratory and cardiac muscles. Since there are no available treatments for cachexia, it is imperative to understand the mechanisms that drive cachexia in order to devise effective strategies to treat it. Although 25% of metastatic breast cancer patients develop symptoms of muscle wasting, mechanistic studies of breast cancer cachexia have been hampered by a lack of experimental models. Using tumor cells deficient for BARD1, a subunit of the BRCA1/BARD1 tumor suppressor complex, we have developed a new orthotopic model of triple-negative breast cancer that spontaneously metastasizes to the lung and leads to systemic muscle deterioration. We show that expression of the metal-ion transporter, Zip14, is markedly upregulated in cachectic muscles from these mice and is associated with elevated intramuscular zinc and iron levels. Aberrant Zip14 expression and altered metal-ion homeostasis could therefore represent an underlying mechanism of cachexia development in human patients with triple-negative breast cancer. Our study provides a unique model for studying breast cancer cachexia and identifies a potential therapeutic target for its treatment.

Programmable bacteria induce durable tumor regression and systemic antitumor immunity.

Synthetic biology is driving a new era of medicine through the genetic programming of living cells1,2. This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics3-6. One particular area of focus has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo7-11. Here we engineered a non-pathogenic Escherichia coli strain to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb)12, an anti-phagocytic receptor that is commonly overexpressed in several human cancer types13,14. We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis and leads to long-term survival in a syngeneic tumor model in mice. Moreover, we report that local injection of CD47nb-expressing bacteria stimulates systemic tumor-antigen-specific immune responses that reduce the growth of untreated tumors, providing proof-of-concept for an abscopal effect induced by an engineered bacterial immunotherapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

Upregulation of ZIP14 and Altered Zinc Homeostasis in Muscles in Pancreatic Cancer Cachexia.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer type in which the mortality rate approaches the incidence rate. More than 85% of PDAC patients experience a profound loss of muscle mass and function, known as cachexia. PDAC patients with this condition suffer from decreased tolerance to anti-cancer therapies and often succumb to premature death due to respiratory and cardiac muscle wasting. Yet, there are no approved therapies available to alleviate cachexia. We previously found that upregulation of the metal ion transporter, Zip14, and altered zinc homeostasis are critical mediators of cachexia in metastatic colon, lung, and breast cancer models. Here, we show that a similar mechanism is likely driving the development of cachexia in PDAC. In two independent experimental metastasis models generated from the murine PDAC cell lines, Pan02 and FC1242, we observed aberrant Zip14 expression and increased zinc ion levels in cachectic muscles. Moreover, in advanced PDAC patients, high levels of ZIP14 in muscles correlated with the presence of cachexia. These studies underscore the importance of altered ZIP14 function in PDAC-associated cachexia development and highlight a potential therapeutic opportunity for improving the quality of life and prolonging survival in PDAC patients.

Metastatic cancers promote cachexia through ZIP14 upregulation in skeletal muscle.

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.

Monitoring Metastasis and Cachexia in a Patient with Breast Cancer: A Case Study.

Cachexia, a wasting syndrome associated with advanced cancer and metastasis, is rarely documented in breast cancer patients. However, the incidence of cachexia in breast cancer is now thought to be largely underestimated. In our case report of a breast cancer patient with bone metastasis monitored during the course of her treatment, we document the development of cachexia by image analysis in relation to her metastatic burden. Elucidation of the link between metastatic burden and cachexia could unveil a highly specific screening process for metastasis, by assessing true muscle mass loss. Our patient was a 49-year-old premenopausal woman, with metastatic invasive ductal breast carcinoma in the vertebral and iliac bones on presentation, which progressed with new metastases to her hips, thigh bones, and vertebrae. In the two-year period, that is between her diagnosis and death, she lost >10% of her baseline weight. During these two years, we retrospectively identified a decrease in paraspinal muscle (PM) at the third lumbar vertebra followed by a sharp decline in weight. The increased tumor burden over time in metastatic sites was accompanied by a decrease in abdominal muscle and visceral and subcutaneous fat and was followed by the patient's demise. The increasing tumor burden in the patient was correlated with the mass of other tissues to determine the tissue that could best serve as a surrogate marker to cachexia and tumor burden. We noted a strong negative correlation between PM area and metastatic tumor area at the third lumbar vertebral level, with PM loss correlating to increasing tumor burden. The monitoring of PM wasting may serve as a marker, and therefore a prognostic factor, for both cachexia and extent of metastatic disease, especially in breast cancer, where metastasis to bone is frequent. Based on our data and review of the literature in this case study, longitudinal monitoring of cachexia in the selected muscle groups can give clinicians early indications of the extent of cachexia in metastatic breast cancer patients.

TNF is a key cytokine mediating neutrophil cytotoxic activity in breast cancer patients.

We have previously shown a novel antimetastatic role for neutrophils in the premetastatic lung of mice in models of breast cancer. Here we expand on those findings in the context of human breast cancer. We assessed the cytotoxicity of neutrophils from 90 newly diagnosed breast cancer patients, 24 ductal carcinoma in situ patients, 56 metastatic breast cancer patients, and 64 women with no history of cancer. We report that neutrophils from metastatic and newly diagnosed breast cancer patients are significantly more cytotoxic than neutrophils from cancer-free individuals. We hypothesized that tumor-secreted factors 'prime' neutrophils to become cytotoxic. To identify these factors we assayed for cytokines in serum from 54 breast cancer patients and 35 cancer-free controls. Tumor necrosis factor (TNFα), MCP-1 (CCL2), and IL1RA significantly correlated with cytotoxicity and directly stimulated neutrophil cytotoxicity ex vivo. RNA-seq analyses found protein kinase C iota (PRKCI) to be over expressed in patient neutrophils relative to neutrophils from cancer-free individuals. PRKCI has been implicated in NADPH oxidase assembly, required for neutrophil-mediated cell cytotoxicity. Treatment of human neutrophils with TNF-induced PRKCI expression and cytotoxicity in samples that had low basal levels of PRKCI expression. To date, this work is the first to demonstrate the cytotoxic role of neutrophils in the peripheral blood of a large cohort of breast cancer patients, and that select cytokines appear to mediate the stimulation of neutrophil cytotoxicity. Further functional studies are necessary to identify clinically relevant means of stimulating neutrophil cytotoxicity as an effective barrier against disease progression and metastasis.

Self-renewal does not predict tumor growth potential in mouse models of high-grade glioma.

Within high-grade gliomas, the precise identities and functional roles of stem-like cells remain unclear. In the normal neurogenic niche, ID (Inhibitor of DNA-binding) genes maintain self-renewal and multipotency of adult neural stem cells. Using PDGF- and KRAS-driven murine models of gliomagenesis, we show that high Id1 expression (Id1(high)) identifies tumor cells with high self-renewal capacity, while low Id1 expression (Id1(low)) identifies tumor cells with proliferative potential but limited self-renewal capacity. Surprisingly, Id1(low) cells generate tumors more rapidly and with higher penetrance than Id1(high) cells. Further, eliminating tumor cell self-renewal through deletion of Id1 has modest effects on animal survival, while knockdown of Olig2 within Id1(low) cells has a significant survival benefit, underscoring the importance of non-self-renewing lineages in disease progression.

Mad2 is a critical mediator of the chromosome instability observed upon Rb and p53 pathway inhibition.

Multiple mechanisms have been proposed to explain how Rb and p53 tumor suppressor loss lead to chromosome instability (CIN). It was recently shown that Rb pathway inhibition causes overexpression of the mitotic checkpoint gene Mad2, but whether Mad2 overexpression is required to generate CIN in this context is unknown. Here, we show that CIN in cultured cells lacking Rb family proteins requires Mad2 upregulation and that this upregulation is also necessary for CIN and tumor progression in vivo. Mad2 is also repressed by p53 and its upregulation is required for CIN in a p53 mutant tumor model. These results demonstrate that Mad2 overexpression is a critical mediator of the CIN observed upon inactivation of two major tumor suppressor pathways.