Targeting Autophagy Addiction in Cancer

Autophagy inhibition is a novel cancer therapeutic strategy in the early stages of clinical trial testing. The initial rationale for using autophagy inhibition was generated by research revealing that autophagy is upregulated in response to external stresses, including chemotherapy and radiotherapy. Combining autophagy inhibition with agents that induce autophagy as a pro-survival response may therefore increase their therapeutic efficacy. Recent research has shown that some cancer cells, particularly those driven by the K-Ras oncogene, also depend on elevated levels of autophagy for survival even in the absence of external stressors. In multiple in vitro as well as in vivo systems, oncogenic Ras-mediated transformation and tumor growth are dependent on autophagy to evade metabolic stress and cell death. These studies have subsequently led to further early phase clinical testing whether autophagy inhibition is a viable and effective strategy for targeting Ras-driven tumors. Even before the clinical results are available from these ongoing clinical trials, much work remains to optimally develop the approach of autophagy inhibition clinically; most notably reliably detecting levels of autophagy in human tumor samples, pharmacodynamics of currently available autophagy inhibitors (chloroquine and the derivative hydroxychloroquine), and new target identification and drug development

NCOA4-Mediated Ferritinophagy: A Potential Link to Neurodegeneration

NCOA4 (Nuclear receptor coactivator 4) mediates the selective autophagic degradation of ferritin, the cellular cytosolic iron storage complex, thereby playing a critical role in intracellular and systemic iron homeostasis. Disruptions in iron homeostasis and autophagy are observed in several neurodegenerative disorders raising the possibility that NCOA4-mediated ferritinophagy links these two observations and may underlie, in part, the pathophysiology of neurodegeneration. Here, we review the available evidence detailing the molecular mechanisms of NCOA4-mediated ferritinophagy and recent studies examining its role in systemic iron homeostasis and erythropoiesis. We propose additional studies to examine the potential role of NCOA4 in the brain in the context of neurodegenerative diseases

Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy

Autophagy, the process by which proteins and organelles are sequestered in double-membrane structures called autophagosomes and delivered to lysosomes for degradation, is critical in diseases such as cancer and neurodegeneration1,2. Much of our understanding of this process has emerged from analysis of bulk cytoplasmic autophagy, but our understanding of how specific cargo including organelles, proteins, or intracellular pathogens are targeted for selective autophagy is limited3. We employed quantitative proteomics to identify a cohort of novel and known autophagosome-enriched proteins, including cargo receptors. Like known cargo receptors, NCOA4 was highly enriched in autophagosomes, and associated with ATG8 proteins that recruit cargo-receptor complexes into autophagosomes. Unbiased identification of NCOA4-associated proteins revealed ferritin heavy and light chains, components of an iron-filled cage structure that protects cells from reactive iron species4 but is degraded via autophagy to release iron5,6 through an unknown mechanism. We found that delivery of ferritin to lysosomes required NCOA4, and an inability of NCOA4-deficient cells to degrade ferritin leads to decreased bioavailable intracellular iron. This work identifies NCOA4 as a selective cargo receptor for autophagic turnover of ferritin (ferritinophagy) critical for iron homeostasis and provides a resource for further dissection of autophagosomal cargo-receptor connectivity

KRAS: feeding pancreatic cancer proliferation

Oncogenic KRAS mutation is the signature genetic event in the progression and growth of pancreatic ductal adenocarcinoma (PDAC), an almost universally fatal disease. Although it has been appreciated for some time that nearly 95% of PDAC harbor mutationally activated KRAS, to date no effective treatments that target this mutant protein have reached the clinic. A number of studies have shown that oncogenic KRAS plays a central role in controlling tumor metabolism by orchestrating multiple metabolic changes including stimulation of glucose uptake, differential channeling of glucose intermediates, reprogrammed glutamine metabolism, increased autophagy, and macropinocytosis. Here we review these recent findings and address how they may be applied to develop new PDAC treatments

Stereotactic Body Radiotherapy (SBRT) Reirradiation for Recurrent Pancreas Cancer

Objectives: After adjuvant or definitive radiation for pancreas cancer, there are limited conventional treatment options for recurrent pancreas cancer. We explored the role of (Stereotactic Body Radiotherapy) SBRT for reirradiation of recurrent pancreas Cancer. Methods: This is a retrospective study of patients reirradiated with SBRT for recurrent pancreas cancer. All patients were deemed unresectable and treated with systemic therapy. Fiducial gold markers were used. CT simulation was performed with oral and IV contrast and patients were treated with respiratory motion tracking in the CyberknifeTM system. Results: 30 patients (17 men and 13 women) with a median age of 67 years were included in the study. The median target volume was 41.29cc. The median prescription dose was 25Gy (24-36Gy) in a median of 5 fractions prescribed to a mean 78% isodose line. The median overall survival was 14 months. The 1 and 2 year local control was 78%. The worst toxicity included 3/30(10%) Grade III acute toxicity for pain, bleeding and vomiting. There was 2/30 (7%) Grade III long-term bowel obstructions. Conclusions: SBRT can be a useful and tolerable option for patients with recurrent pancreas cancer after prior radiation

An In Vivo CRISPR Screening Platform for Prioritizing Therapeutic Targets in AML

CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275

Neoadjuvant Chemotherapy Is Associated with Altered Immune Cell Infiltration and an Anti-Tumorigenic Microenvironment in Resected Pancreatic Cancer

PURPOSE: Neoadjuvant chemotherapy is increasingly administered to patients with resectable or borderline resectable pancreatic ductal adenocarcinoma (PDAC), yet its impact on the tumor immune microenvironment is incompletely understood. DESIGN: We employed quantitative, spatially resolved multiplex immunofluorescence and digital image analysis to identify T-cell subpopulations, macrophage polarization states, and myeloid cell subpopulations in a multi-institution cohort of up-front resected primary tumors (n = 299) and in a comparative set of resected tumors after FOLFIRINOX-based neoadjuvant therapy (n = 36) or up-front surgery (n = 30). Multivariable-adjusted Cox proportional hazards models were used to evaluate associations between the immune microenvironment and patient outcomes. RESULTS: In the multi-institutional resection cohort, immune cells exhibited substantial heterogeneity across patient tumors and were located predominantly in stromal regions. Unsupervised clustering using immune cell densities identified four main patterns of immune cell infiltration. One pattern, seen in 20% of tumors and characterized by abundant T cells (T cell-rich) and a paucity of immunosuppressive granulocytes and macrophages, was associated with improved patient survival. Neoadjuvant chemotherapy was associated with a higher CD8:CD4 ratio, greater M1:M2-polarized macrophage ratio, and reduced CD15+ARG1+ immunosuppressive granulocyte density. Within neoadjuvant-treated tumors, 72% showed a T cell-rich pattern with low immunosuppressive granulocytes and macrophages. M1-polarized macrophages were located closer to tumor cells after neoadjuvant chemotherapy, and colocalization of M1-polarized macrophages and tumor cells was associated with greater tumor pathologic response and improved patient survival. CONCLUSIONS: Neoadjuvant chemotherapy with FOLFIRINOX shifts the PDAC immune microenvironment toward an anti-tumorigenic state associated with improved patient survival

Spatially Resolved Single-Cell Assessment of Pancreatic Cancer Expression Subtypes Reveals Co-expressor Phenotypes and Extensive Intratumoral Heterogeneity

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in \u3e 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell-cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field