Role of NuMA1 in breast cancer stem cells with implications for combination therapy of PIM1 and autophagy inhibition in triple negative breast cancer.

Background: Nuclear mitotic apparatus protein 1 (NuMA1) is a cell cycle protein and upregulated in breast cancer. However, the role of NuMA1 in TNBC and its regulation in heterogenous populations remains elusive. Methods: We performed CRISPR mediated deletion of NuMA1 in mouse TNBC cells, BF3M. FACS was utilized to isolate BCSCs, and bulk cells based on CD29 and CD61 markers. Cell viability, migration, and invasion ability of BCSCs and bulk cells was evaluated using MTT, wound healing and transwell invasion assays, respectively. In vivo mouse breast cancer and lung metastatic models were generated to evaluate the combination treatment of SMI-4a and Lys-o5 inhibitors. Results: We identified that high expression of NuMA1 associated with poor survival of breast cancer patients. Further, human tissue microarray results depicted high expression of NuMA1 in TNBC relative to non-adjacent normal tissues. Therefore, we performed CRISPR mediated deletion of NuMA1 in a mouse mammary tumor cell line, BF3M and revealed that NuMA1 deletion reduced mammary tumorigenesis. We also showed that NuMA1 deletion reduced ALDH+ and CD29hiCD61+ breast cancer stem cells (BCSCs), indicating a role of NuMA1 in BCSCs. Further, sorted and characterized BCSCs from BF3M depicted reduced metastasis with NuMA1 KO cells. Moreover, we found that PIM1, an upstream kinase of NuMA1 plays a preferential role in maintenance of BCSCs associated phenotypes, but not in bulk cells. In contrast, PIM1 kinase inhibition in bulk cells depicted increased autophagy (FIP200). Therefore, we applied a combination treatment strategy of PIM1 and autophagy inhibition using SMI-4a and Lys05 respectively, showed higher efficacy against cell viability of both these populations and further reduced breast tumor formation and metastasis. Together, our study demonstrated NuMA1 as a potential therapeutic target and combination treatment using inhibitors for an upstream kinase PIM1 and autophagy inhibitors could be a potentially new therapeutic approach for TNBC. Conclusions: Our study demonstrated that combination treatment of PIM1 inhibitor and autophagy inhibitor depicted reduced mammary tumorigenesis and metastasis by targeting NuMA1 in BCSCs and bulk cells of TNBC, demonstrating this combination treatment approach could be a potentially effective therapy for TNBC patients.

In vivo CRISPR knockout screen identifies p47 as a suppressor of HER2+ breast cancer metastasis by regulating NEMO trafficking and autophagy flux.

Autophagy is a conserved cellular process, and its dysfunction is implicated in cancer and other diseases. Here, we employ an in vivo CRISPR screen targeting genes implicated in the regulation of autophagy to identify the Nsfl1c gene encoding p47 as a suppressor of human epidermal growth factor receptor 2 (HER2)+ breast cancer metastasis. p47 ablation specifically increases metastasis without promoting primary mammary tumor growth. Analysis of human breast cancer patient databases and tissue samples indicates a correlation of lower p47 expression levels with metastasis and decreased survival. Mechanistic studies show that p47 functions in the repair of lysosomal damage for autophagy flux and in the endosomal trafficking of nuclear factor κB essential modulator for lysosomal degradation to promote metastasis. Our results demonstrate a role and mechanisms of p47 in the regulation of breast cancer metastasis. They highlight the potential to exploit p47 as a suppressor of metastasis through multiple pathways in HER2+ breast cancer cells.

AZI2 mediates TBK1 activation at unresolved selective autophagy cargo receptor complexes with implications for CD8 T-cell infiltration in breast cancer.

Most breast cancers do not respond to immune checkpoint inhibitors and there is an urgent need to identify novel sensitization strategies. Herein, we uncovered that activation of the TBK-IFN pathway that is mediated by the TBK1 adapter protein AZI2 is a potent strategy for this purpose. Our initial observations showed that RB1CC1 depletion leads to accumulation of AZI2, in puncta along with selective macroautophagy/autophagy cargo receptors, which are both required for TBK1 activation. Specifically, disrupting the selective autophagy function of RB1CC1 was sufficient to sustain AZI2 puncta accumulation and TBK1 activation. AZI2 then mediates downstream activation of DDX3X, increasing its interaction with IRF3 for transcription of pro-inflammatory chemokines. Consequently, we performed a screen to identify inhibitors that can induce the AZI2-TBK1 pathway, and this revealed Lys05 as a pharmacological agent that induced pro-inflammatory chemokine expression and CD8+ T cell infiltration into tumors. Overall, we have identified a distinct AZI2-TBK1-IFN signaling pathway that is responsive to selective autophagy blockade and can be activated to make breast cancers more immunogenic.Abbreviations: AZI2/NAP1: 5-azacytidine induced 2; CALCOCO2: calcium binding and coiled-coil domain 2; DDX3X: DEAD-box helicase 3 X-linked; FCCP: carbonyl cyanide p-triflouromethoxyphenylhydrazone; a protonophore that depolarizes the mitochondrial inner membrane; ICI: immune checkpoint inhibitor; IFN: interferon; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1.

Regulation of RB1CC1/FIP200 stability and autophagy function by CREBBP-mediated acetylation in an intrinsically disordered region.

RB1CC1/FIP200 is an essential macroautophagy/autophagy protein that plays an important role in a variety of biological and disease processes through its canonical autophagy-dependent and -independent functions. However, it remains largely unknown whether post-translational modifications could regulate RB1CC1 and its associated autophagy functions. Here, we report acetylation of several lysine residues of RB1CC1 by acetyltransferase CREBBP (CREB binding protein), with K276 as the major CREBBP acetylation site. K276 is also identified as a ubiquitination site by mass spectrometry, and acetylation at this site reduces ubiquitination of RB1CC1 to inhibit its ubiquitin-dependent degradation. We also find that RB1CC1 contains an N-terminal intrinsically disordered region (IDR) capable of forming liquid-liquid phase separation (LLPS) in vitro, which may drive formation of RB1CC1 puncta with LLPS properties in cells independent of SQSTM1/p62 and other autophagy receptors CALCOCO2/NDP52, NBR1, TAX1BP1 and OPTN. Mutational analysis shows that both K276 acetylation and the N-terminal IDR containing it are important for maintaining canonical autophagy function of RB1CC1 in breast cancer cells. Our findings demonstrate regulation of RB1CC1 by a new post-translational mechanism and suggest potential therapeutic application of inducing RB1CC1 degradation through blocking K276 acetylation in the treatment of cancer and other diseases.Abbreviations: Baf-A1: bafilomycin A1; CREBBP/CBP: CREB binding protein; CHX: cycloheximide; EP300/p300: E1A binding protein p300; FRAP: fluorescence recovery after photobleaching; HADCs: histone deacetylases; IDR: intrinsically disordered region; LLPS: liquid-liquid phase separation; KAT2A/GCN5: lysine acetyltransferase 2A; KAT2B/PCAF: lysine acetyltransferase 2B; KAT5/TIP60: lysine acetyltransferase 5; KAT8/MOF: lysine acetyltransferase 8; NAM: nicotinamide; PAS: phagophore assembly site; PEG-8000: polyethylene glycol 8000; RB1CC1/FIP200: RB1 inducible coiled-coil 1; TSA: trichostatin A.

Targeting Autophagy in Thyroid Cancer: EMT, Apoptosis, and Cancer Stem Cells.

Autophagy is a highly conserved recycling process through which cellular homeostasis is achieved and maintained. With respect to cancer biology, autophagy acts as a double-edged sword supporting tumor cells during times of metabolic and therapeutic stress, while also inhibiting tumor development by promoting genomic stability. Accumulating evidence suggests that autophagy plays a role in thyroid cancer, acting to promote tumor cell viability and metastatic disease through maintenance of cancer stem cells (CSCs), supporting epithelial-to-mesenchymal transition (EMT), and preventing tumor cell death. Intriguingly, well-differentiated thyroid cancer is more prevalent in women as compared to men, though the underlying molecular biology driving this disparity has not yet been elucidated. Several studies have demonstrated that autophagy inhibitors may augment the anti-cancer effects of known thyroid cancer therapies. Autophagy modulation has become an attractive target for improving outcomes in thyroid cancer. This review aims to provide a comprehensive picture of the current knowledge regarding the role of autophagy in thyroid cancer, focusing on the potential mechanism(s) through which inhibition of autophagy may enhance cancer therapy and outcomes.

Biglycan Promotes Cancer Stem Cell Properties, NFκB Signaling and Metastatic Potential in Breast Cancer Cells.

It is a major challenge to treat metastasis due to the presence of heterogenous BCSCs. Therefore, it is important to identify new molecular targets and their underlying molecular mechanisms in various BCSCs to improve treatment of breast cancer metastasis. Here, we performed RNA sequencing on two distinct co-existing BCSC populations, ALDH+ and CD29hi CD61+ from PyMT mammary tumor cells and detected upregulation of biglycan (BGN) in these BCSCs. Genetic depletion of BGN reduced BCSC proportions and tumorsphere formation. Furthermore, BCSC associated aggressive traits such as migration and invasion were significantly reduced by depletion of BGN. Glycolytic and mitochondrial metabolic assays also revealed that BCSCs exhibited decreased metabolism upon loss of BGN. BCSCs showed decreased activation of the NFκB transcription factor, p65, and phospho-IκB levels upon BGN ablation, indicating regulation of NFκB pathway by BGN. To further support our data, we also characterized CD24-/CD44+ BCSCs from human luminal MCF-7 breast cancer cells. These CD24-/CD44+ BCSCs similarly exhibited reduced tumorigenic phenotypes, metabolism and attenuation of NFκB pathway after knockdown of BGN. Finally, loss of BGN in ALDH+ and CD29hi CD61+ BCSCs showed decreased metastatic potential, suggesting BGN serves as an important therapeutic target in BCSCs for treating metastasis of breast cancer.

Enhanced autophagy in Becn1F121A/F121A knockin mice counteracts aging-related neural stem cell exhaustion and dysfunction.

Macroautophagy/autophagy is emerging as a major pathway that regulates both aging and stem cell function. Previous studies have demonstrated a positive correlation of autophagy with longevity; however, these studies did not directly address the consequence of altered autophagy in stem cells during aging. In this study, we used Becn1F121A/F121A knockin mice (designated as Becn1 KI mice) with the F121A allele in the autophagy gene Becn1 to investigate the consequences of enhanced autophagy in postnatal neural stem cells (NSCs) during aging. We found that increased autophagy protected NSCs from exhaustion and promoted neurogenesis in old (≥18-months-old) mice compared with age-matched wild-type (WT) mice, although it did not affect NSCs in young (3-months-old) mice. After pharmacologically-induced elimination of proliferative cells in the subventricular zone (SVZ), there was enhanced re-activation of quiescent NSCs in old Becn1 KI mice as compared to those in WT mice, with more efficient exit from quiescent status to generate proliferative cells and neuroblasts. Moreover, there was also improved maintenance and increased neuronal differentiation of NSCs isolated from the SVZ of old Becn1 KI mice in in vitro assays. Lastly, the increased neurogenesis in Becn1 KI mice was associated with better olfactory function in aged animals. Together, our results suggest a protective role of increased autophagy in aging NSCs, which may help the development of novel strategies to treat age-related neurodegeneration.Abbreviations: ATG: autophagy related; Baf A1: bafilomycin A1; Becn1: beclin 1; BrdU: bromodeoxyuridine/5-bromo-2'-deoxyuridine; DCX: doublecortin; GFAP: glial fibrillary acidic protein; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSCs: hematopoietic stem cells; KI: knockin; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mo: month; NSCs: neural stem cells; OB: olfactory bulb; RB1CC1: RB1-inducible coiled-coil 1; ROS: reactive oxygen species; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; TMZ: temozolomide; WT: wild type.

Non-canonical function of FIP200 is required for neural stem cell maintenance and differentiation by limiting TBK1 activation and p62 aggregate formation.

FIP200 is an essential autophagy gene implicated in the regulation of postnatal neural progenitor/stem cells (NSCs). However, the contribution of FIP200's canonical-autophagy function and its non-canonical functions to postnatal NSC maintenance remains unclear. Utilizing a recently generated Fip200-4A allele that specifically impairs FIP200's canonical-autophagy function, we found that non-canonical functions of FIP200 was required for regulation of mouse NSC maintenance and neurogenesis in vivo. Ablating the non-canonical functions of FIP200, but not its autophagy function, increased TBK1 activation and p62 phosphorylation at S403 in NSCs. Phosphorylation of p62 was dependent on TBK1 kinase activity and increased the propensity of p62 aggregate formation specifically in FIP200-null NSCs. Accordingly, inhibition of TBK1 by amlexanox reduced p62 aggregates and restored NSC maintenance and differentiation in Fip200hGFAP cKO mice. These results reveal a mechanism for the non-canonical functions of FIP200 in NSC maintenance and differentiation by limiting TBK1 activation and subsequently, p62 aggregate formation.

Heterogeneity within molecular subtypes of breast cancer.

Breast cancer is the quintessential example of how molecular characterization of tumor biology guides therapeutic decisions. From the discovery of the estrogen receptor to current clinical molecular profiles to evolving single-cell analytics, the characterization and compartmentalization of breast cancer into divergent subtypes is clear. However, competing with this divergent model of breast cancer is the recognition of intratumoral heterogeneity, which acknowledges the possibility that multiple different subtypes exist within a single tumor. Intratumoral heterogeneity is driven by both intrinsic effects of the tumor cells themselves as well as extrinsic effects from the surrounding microenvironment. There is emerging evidence that these intratumoral molecular subtypes are not static; rather, plasticity between divergent subtypes is possible. Interconversion between seemingly different subtypes within a tumor drives tumor progression, metastases, and treatment resistance. Therapeutic strategies must, therefore, contend with changing phenotypes in an individual patient's tumor. Identifying targetable drivers of molecular heterogeneity may improve treatment durability and disease progression.

Autophagy inhibition perturbs ERBB2 trafficking and abolishes tumorigenesis in ERBB2-driven breast cancer.

Macroautophagy/autophagy modulation is increasingly recognized as a potential strategy for cancer therapy. Using a recently developed Rb1cc1 mutant knockin mice model, we have taken a rigorous genetic approach to assess the role of both its autophagy and non-canonical functions in an ERBB2-driven BrCA model. We found that autophagy abrogation virtually abolishes mammary tumorigenesis in the ERBB2-driven model, exhibiting stronger inhibitory effects than in our previous studies using PyMT and brca1-null mouse models. Mechanistically, autophagy inhibition perturbs ERBB2 intracellular trafficking and triggers its release via small extracellular vesicles. Our results demonstrate a new mechanism for autophagy to promote tumorigenesis in ERBB2-driven BrCA and could supplement current strategies for anti-ERBB2 therapy.

Functional cooperation between co-amplified genes promotes aggressive phenotypes of HER2-positive breast cancer.

MED1 (mediator subunit 1) co-amplifies with HER2, but its role in HER2-driven mammary tumorigenesis is still unknown. Here, we generate MED1 mammary-specific overexpression mice and cross them with mouse mammary tumor virus (MMTV)-HER2 mice. We observe significantly promoted onset, growth, metastasis, and multiplicity of HER2 tumors by MED1 overexpression. Further studies reveal critical roles for MED1 in epithelial-mesenchymal transition, cancer stem cell formation, and response to anti-HER2 therapy. Mechanistically, RNA sequencing (RNA-seq) transcriptome analyses and clinical sample correlation studies identify Jab1, a component of the COP9 signalosome complex, as the key direct target gene of MED1 contributing to these processes. Further studies reveal that Jab1 can also reciprocally regulate the stability and transcriptional activity of MED1. Together, our findings support a functional cooperation between these co-amplified genes in HER2+ mammary tumorigenesis and their potential usage as therapeutic targets for the treatment of HER2+ breast cancers.

Autophagy mediated lipid catabolism facilitates glioma progression to overcome bioenergetic crisis.

BACKGROUND: Activation of mTORC1 plays a significant role in cancer development and progression. However, the metabolic mechanisms to sustain mTORC1 activation of cancer cells within stressed environments are still under-appreciated. We recently revealed high autophagy activity in tumour cells with mTORC1 hyper-activation. Nevertheless, the functions and mechanisms of autophagy in regulating mTORC1 in glioma are not studied. METHODS: Using glioma patient database and human glioma cells, we assessed the mechanisms and function of selective autophagy to sustain mTORC1 hyper-activation in glioma. RESULTS: We revealed a strong association of altered mRNA levels in mTORC1 upstream and downstream genes with prognosis of glioma patients. Our results indicated that autophagy-mediated lipid catabolism was essential to sustain mTORC1 activity in glioma cells under energy stresses. We found that autophagy inhibitors or fatty acid oxidation (FAO) inhibitors in combination with 2-Deoxy-D-glucose (2DG) decreased energy production and survival of glioma cells in vitro. Consistently, inhibition of autophagy or FAO inhibitors with 2DG effectively suppressed the progression of xenografted glioma with hyper-activated mTORC1. CONCLUSIONS: This study established an autophagy/lipid degradation/FAO/ATP generation pathway, which might be used in brain cancer cells under energy stresses to maintain high mTORC1 signalling for tumour progression.

Autophagy Blockade Limits HER2+ Breast Cancer Tumorigenesis by Perturbing HER2 Trafficking and Promoting Release Via Small Extracellular Vesicles.

Autophagy modulation is an emerging strategy for cancer therapy. By deleting an essential autophagy gene or disrupting its autophagy function, we determined a mechanism of HER2+ breast cancer tumorigenesis by directly regulating the oncogenic driver. Disruption of FIP200-mediated autophagy reduced HER2 expression on the tumor cell surface and abolished mammary tumorigenesis in MMTV-Neu mice. Decreased HER2 surface expression was due to trafficking from the Golgi to the endocytic pathways instead of the plasma membrane. Autophagy inhibition led to HER2 accumulation in early and late endosomes associated with intraluminal vesicles and released from tumor cells in small extracellular vesicles (sEVs). Increased HER2 release from sEVs correlated with reduced tumor cell surface levels. Blocking sEVs secretion rescued HER2 levels in tumor cells. Our results demonstrate a role for autophagy to promote tumorigenesis in HER2+ breast cancer. This suggests that blocking autophagy could supplement current anti-HER2 agents for treating the disease.

Single-cell RNA-sequencing reveals distinct patterns of cell state heterogeneity in mouse models of breast cancer.

Breast cancer stem cells (BCSCs) contribute to intra-tumoral heterogeneity and therapeutic resistance. However, the binary concept of universal BCSCs co-existing with bulk tumor cells is over-simplified. Through single-cell RNA-sequencing, we found that Neu, PyMT and BRCA1-null mammary tumors each corresponded to a spectrum of minimally overlapping cell differentiation states without a universal BCSC population. Instead, our analyses revealed that these tumors contained distinct lineage-specific tumor propagating cells (TPCs) and this is reflective of the self-sustaining capabilities of lineage-specific stem/progenitor cells in the mammary epithelial hierarchy. By understanding the respective tumor hierarchies, we were able to identify CD14 as a TPC marker in the Neu tumor. Additionally, single-cell breast cancer subtype stratification revealed the co-existence of multiple breast cancer subtypes within tumors. Collectively, our findings emphasize the need to account for lineage-specific TPCs and the hierarchical composition within breast tumors, as these heterogenous sub-populations can have differential therapeutic susceptibilities.

Regulation of immune checkpoint blockade efficacy in breast cancer by FIP200: A canonical-autophagy-independent function.

Immune checkpoint blockade (ICB) has emerged as a promising therapeutic strategy because of its potential to induce durable therapeutic responses in cancer patients. However, in the case of breast cancer, its application and efficacy has been limited. As such, combinatorial therapeutic strategies that can unlock the potential of ICB in breast cancer are of urgent need. In view of that, autophagy-related proteins that play a role in the autophagic cell recycling process have been implicated in the regulation of inflammatory and anti-tumor immune responses. Accordingly, autophagy-related proteins represent a group of prospective therapeutic targets in conjunction with ICB. In our recent study (Okamoto T et al. (2020), Cancer Res), we developed immune-competent mouse models of breast cancer which were deficient for the autophagic function of FIP200 or had FIP200 completely ablated to test the efficacy of ICB. We showed that although FIP200's autophagy function was required for progression of PyMT-driven mammary tumors, FIP200's canonical-autophagy-independent function was responsible for increased T-cell infiltration, IFN-signaling and ICB efficacy. These findings provide genetic proof of principle for a combinatorial therapeutic strategy that involves ablation of FIP200 to improve ICB efficacy in non-responsive breast cancers.

Role of FIP200 in inflammatory processes beyond its canonical autophagy function.

FIP200 (RB1CC1) is a critical regulator of canonical macroautophagy and has also emerged as a crucial regulator of selective autophagy as well as inflammatory processes. The illumination of FIP200's role in autophagy at the molecular level has been accompanied by studies demonstrating the importance of its autophagy function in physiological processes in mammals and pathological contexts such as cancer. However, there is an increasing appreciation that most, if not all of the autophagy genes, also play a role in other processes such as LC3-associated phagocytosis, vesicle trafficking and protein secretion. Consequently, this has led to efforts in generating specific mutants of autophagy genes that are more amenable to dissecting their autophagy versus non-autophagy functions. In this aspect, we have generated a FIP200 knock-in mouse allele that is defective for canonical macroautophagy. This has revealed a canonical-autophagy-independent function of FIP200 that is responsible for limiting pro-inflammatory signaling. In this review, we will discuss FIP200's role in this process, the implications with regards to cancer immunotherapy and highlight key prospective avenues to specifically dissect the distinct functions of FIP200.

Correction to: FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors.

An amendment to this paper has been published and can be accessed via the original article.

FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors.

BACKGROUND: Breast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer. METHODS: To understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested. RESULTS: We found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival. CONCLUSIONS: In summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

FIP200 Suppresses Immune Checkpoint Therapy Responses in Breast Cancers by Limiting AZI2/TBK1/IRF Signaling Independent of Its Canonical Autophagy Function.

Immune checkpoint inhibitors (ICI) have the potential to induce durable therapeutic responses, yet response rates in breast cancer are modest and limited to particular subtypes. To expand the applicability of ICI, we examined the role of an essential autophagy gene, FIP200, which has been shown to be important for tumor progression in mammary tumors. Specific disruption of the autophagy function of FIP200 or complete ablation of FIP200 in genetic mouse models revealed that FIP200 autophagy function was required for progression of PyMT-driven mammary tumors. However, a noncanonical autophagy function of FIP200 was responsible for limiting T-cell recruitment and activation of the TBK1-IFN signaling axis. FIP200 also interacted with the TBK1 adaptor protein, AZI2, which was crucial for activation of TBK1 following FIP200 ablation. Accordingly, disrupting the noncanonical autophagy function of FIP200 in combination with ICI therapy led to superior, durable responses in immune-competent models of breast cancer. Collectively, these insights could guide future development of therapeutic agents against FIP200 for combinatorial ICI therapies in nonresponsive breast cancers. SIGNIFICANCE: These findings show that deletion of FIP200 enhances immune checkpoint inhibitor efficacy in nonresponsive breast cancer.

Targeted therapy for mTORC1-driven tumours through HDAC inhibition by exploiting innate vulnerability of mTORC1 hyper-activation.

BACKGOUND: The mechanistic target of rapamycin complex 1 (mTORC1) is important in the development and progression of many cancers. Targeted cancer therapy using mTORC1 inhibitors is used for treatment of cancers; however, their clinical efficacies are still limited. METHODS: We recently created a new mouse model for human lymphangiosarcoma by deleting Tsc1 in endothelial cells and consequent hyper-activation of mTORC1. Using Tsc1iΔEC tumour cells from this mouse model, we assessed the efficacies of histone deacetylase (HDAC) inhibitors as anti-tumour agents for mTORC1-driven tumours. RESULTS: Unlike the cytostatic effect of mTORC1 inhibitors, HDAC inhibitors induced Tsc1iΔEC tumour cell death in vitro and their growth in vivo. Analysis of several HDAC inhibitors suggested stronger anti-tumour activity of class I HDAC inhibitor than class IIa or class IIb inhibitors, but these or pan HDAC inhibitor SAHA did not affect mTORC1 activation in these cells. Moreover, HDAC inhibitor-induced cell death required elevated autophagy, but was not affected by disrupting caspase-dependent apoptosis pathways. We also observed increased reactive oxygen species and endoplasmic reticulum stress in SAHA-treated tumour cells, suggesting their contribution to autophagic cell death, which were dependent on mTORC1 hyper-activation. CONCLUSION: These studies suggest a potential new treatment strategy for mTORC1-driven cancers like lymphangiosarcoma through an alternative mechanism.