Supplementary Data from Type I but Not Type II Calreticulin Mutations Activate the IRE1α/XBP1 Pathway of the Unfolded Protein Response to Drive Myeloproliferative Neoplasms

We and others previously showed that extracellular ATP (eATP) is implicated in epithelial mesenchymal transition (EMT). However, the mechanisms by which eATP induces EMT and ATP’s relationship to TGF-β, a well-known EMT inducer, are largely unclear. Also, eATP-induced EMT has never been studied at transcriptomic and metabolomics levels. Based on our previous studies, we hypothesized that eATP acts as a specific inducer and regulator of EMT at all levels in cancer cells. RNAseq and metabolomics analyses were performed on human non-small cell lung cancer (NSCLC) A549 cells treated with either eATP or TGF-β. Bio-functional assays, such as invasion, intracellular ATP, cell proliferation, cytoskeleton remodeling, and others were conducted in NSCLC A549 and H1299 cells to validate changes observed from RNAseq and metabolomics studies. In the RNAseq study, eATP significantly enriched expressions of genes involved in EMT similarly to TGF-β after 2 and 6 hours of treatment. Samples treated with eATP for 2 hours share 131 upregulated EMT genes with those of TGF-β treated samples, and 42 genes at 6 hours treatment. Eleven genes, with known or unknown functions in EMT, are significantly upregulated by both inducers at both time points, have been identified. BLOC1S6, one of the 11 genes, was selected for further study. eATP induced numerous EMT-related changes in metabolic pathways, including cytoskeleton rearrangement, glycolysis, glutaminolysis, ROS, and individual metabolic changes similar to those induced by TGF-β. Functional bioassays verified the findings from RNAseq and metabolomics that eATP EMT-like changes in A549 and H1299 cells similarly to TGF-β. BLOC1S6 was found to be implicated in EMT. In these studies, eATP-induced EMT, at all levels examined, is similar but non-identical to that induced by TGF-β, and functions in such a way that exogenous addition of TGF-β is unnecessary for the induction. The study of BLOC1S6 further verified its potential roles in EMT and the RNAseq analysis results. All these strongly indicate that eATP is a multi-functional and multi-locational inducer and regulator of EMT, changing our thinking on how EMT is induced and regulated and pointing to new directions for inhibiting EMT in cancer.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain. Here, we show that type 1 CALRdel52 but not type 2 CALRins5 mutations lead to activation of and dependency on the IRE1α-XBP1 pathway of the unfolded protein response (UPR). Mechanistically, we found that the loss of calcium binding residues in the type 1 mutant CALR protein directly impairs its calcium binding ability, which in turn leads to depleted ER calcium and subsequent activation of the IRE1α-XBP1 pathway. Using cell lines and primary MPN patient samples, we identified two novel transcriptional targets of XBP1 specific to CALRdel52-expressing cells - the anti-apoptotic protein BCL-2 and the calcium efflux channel IP3R. We show that BCL-2 acts downstream of XBP1 to promote survival in the face of depleted ER calcium, while IP3R is up-regulated downstream of XBP1 to promote continued ER calcium efflux in order to sustain IRE1α-XBP1 pathway activation and survival. We found that genetic or pharmacological inhibition of IRE1α-XBP1 signaling induced cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells. Moreover, we show that in vivo inhibition of IRE1α significantly abrogates type 1 mutant CALR-driven disease in a bone marrow transplantation model. This work is the first to demonstrate that type 1 and type 2 mutant CALR-expressing cells display differential molecular dependencies that can be exploited for therapeutic gain. Moreover, this study answers an enduring question regarding the functional consequence of the loss of calcium binding sites on the type 1 mutant CALR protein, and demonstrates how type 1 CALR mutant-expressing cells rewire the UPR, downstream calcium signaling, and apoptotic pathways to drive MPN. Citation Format: Juan Ibarra, Yassmin Elbanna, Katarzyna Kurylowicz, Michele Ciboddo, Harrison S. Greenbaum, Nicole S. Arellano, Deborah Rodriguez, Maria Evers, Dongbo Yang, Althea Bock-Hughes, Chenyu Liu, Quinn Smith, Julian Baumeister, Milena Kalmer, Kathrin Olschok, Benjamin Nicholson, Diane Silva, Jonathan Dowgielewicz3, Elisa Rumi, Daniela Pietra, Ilaria Carola Casetti, Steffen Koschmieder5, Sandeep Gurbuxani, Rebekka K. Schneider, Scott A. Oakes, Shannon E. Elf. Type 1 calreticulin mutations differentially activate the IRE1α-XBP1 pathway of the unfolded protein response to drive myeloproliferative neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB134.

Cancer stem cells (CSCs) are closely associated with metastasis and epithelial mesenchymal transition (EMT). We previously reported that extracellular ATP (eATP) induces and regulates EMT in cancer cells. We recently found that the gene stanniocalcin 1 (STC1) is significantly upregulated by eATP in human non-small lung cancer (NSCLC) A549 cells; however, the relationships among eATP, CSCs, and STC1 were largely unknown. In this study, we performed gene knockdown and knockout, and a wide variety of functional assays to determine if and how eATP and STC1 induce CSCs in NSCLC A549 and H1299 cells. Our data show that, in both cultured cells and tumors, eATP increased the number of CSCs in the cancer cell population and upregulated CSC-related genes and protein markers. STC1 deletion led to drastically slower cell and tumor growth, reduced intracellular ATP levels and CSC markers, and metabolically shifted STC1-deficient cells from an energetic state to a quiescent state. These findings indicate that eATP induces and regulates CSCs at transcriptional, translational, and metabolic levels, and these activities are mediated through STC1 via mitochondria-associated ATP synthesis. These novel findings offer insights into eATP-induced CSCs and identify new targets for inhibiting CSCs.

Macropinocytosis is one of the major mechanisms by which cancer cells uptake extracellular nutrients from tumor microenvironment (TME) and plays very important roles in various steps of tumorigenesis. We previously reported the unexpected finding that intratumoral and extracellular ATP (eATP), as one of the major drastically upregulated extracellular nutrients and messengers in tumors, is taken up by cancer cells through macropinocytosis in large quantities and significantly contributing to cancer cell growth, survival, and increased resistance to chemo and target drugs. Inhibition of macropinocytosis substantially reduced eATP uptake by cancer cells and slowed down tumor growth in vivo. More recently, we have found the eATP also plays a very important role in inducing epithelial-to-mesenchymal transition (EMT), and that macropinocytosis is an essential facilitator in the induction. Thus, macropinocytosis and eATP, working in coordination, appear to play some previously unrecognized but very important roles in EMT and metastasis. As a result, they are likely to be interactive and communicative with each other, regulating each other's activity for various needs of host tumor cells. They are also likely to be an integral part of the future new anticancer therapeutic strategies. Moreover, it is undoubted that we have not identified all the important activities coordinated by ATP and macropinocytosis. This review describes our findings in how eATP and macropinocytosis work together to promote cancer cell growth, resistance, and EMT. We also list scientific challenges facing eATP research and propose to target macropinocytosis and eATP to reduce drug resistance and slow down metastasis.

Abstract Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. Significance: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type–specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). 80% of CALR mutations are classified as either type 1 or type 2, exemplified by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5), respectively. Despite their shared mutant C-termini and mutual ability to bind and activate MPL, patients with type 1 and type 2 CALR mutations display significant clinical and prognostic differences. Type 1 mutations are primarily associated with an MF phenotype and a higher risk of fibrotic transformation from ET, while type 2 mutations are more common in ET. Molecularly, type 2 CALR mutant proteins retain many of the calcium binding sites present in the wild type protein, while type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of calcium binding sites remain yet unexplored. Current targeted therapies for CALR mutated MPN are not curative, and treatment does not differentiate between type 1 versus type 2 mutant CALR-driven disease, despite the different phenotypic and prognostic outcomes in these patients. In order to improve treatment strategies for CALR mutated MPN patients, it is critical to identify specific dependencies unique to each CALR mutation type that can be exploited for therapeutic gain.

Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). Of these, approximately half are classified as type 1 and 30% as type 2, characterized by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5) respectively. Although both share identical mutant C-termini and are able to bind and activate MPL, type 1 and type 2 CALR mutations display different clinical and prognostic presentation: type 1 mutations are associated primarily with a fibrotic phenotype and increased proclivity towards fibrotic transformation, while type 2 mutations are more common in ET. Molecularly, type 1 and type 2 mutations result in differential C-domain amino acid sequences with the potential to affect the function of the protein. Various well known functions of CALR, including calcium binding ability and protein folding capacity, have begun to be explored in the context of CALR mutations; however, the impact of CALR mutations on its acetyltransferase ability, which was only discovered in 2006, remains unknown.

Abstract Cancer metastasis, the spread of cancer to new places in the body, is associated with ~90% of solid tumor related deaths, yet remains one of the least understood processes in cancer. Epithelial-mesenchymal transition (EMT) is one of the first initiating steps that is necessary for cancer metastasis. During EMT, cancer cells lose their cell-cell adhesion and cell polarity, and gain migratory and invasive properties in preparation for metastasis. During this process, cells lose markers and transcription factors associated with the epithelial state, and gain markers and transcription factors associated with the mesenchymal state. TGF-β, a cytokine, has long been known to be an inducer for EMT in cancer. In addition to TGF-β, extracellular ATP (eATP) has recently emerged to be an alternative EMT inducer that has important functions involved in inducing EMT and EMT-related processes such as drug resistance [1]. Intratumoral extracellular ATP levels are to 103 to 104 times higher than those in normal tissues (in the high μM range), suggesting its biological importance. Considering this, we hypothesize that ATP and TGF-β may be redundant and complimentary molecules in the induction of EMT, providing cancer the flexibility to use whatever is present at the time. However, the complex functional relationship between eATP and TGF-β has not previously been investigated. We seek to identify and elucidate the mechanisms and relationships by which ATP and TGF-β induce EMT/metastasis. We have found that, in A549 human non-small cell lung cancer cells, ATP and TGF-β are able to induce comparable amounts of cancer cell migration and invasion using in vitro transwell assays. Total RNAseq and metabolomics studies will be completed to identify the transcriptional and metabolic changes associated with the ATP or TGF-β -activated EMT process. A comparative study of the effects of ATP and TGF-β in the early steps of metastasis have never before been reported, leaving uncertainty in their relationship to each other and their necessity and sufficiency in the EMT process. The elucidation of ATP's mechanisms of action in the processes will significantly enhance our understanding of ATP, cancer, and metastasis, eventually leading to novel and more effective ways of slowing down metastasis and reducing metastasis-related deaths. [1] Cao Y, Wang X, Li Y, Evers M, Zhang H, Chen X. (2019). Extracellular and macropinocytosis internalized ATP work together to induce epithelial-mesenchymal transition and other early metastatic activities in lung cancer. Cancer Cell Int. Citation Format: Maria D. Evers, Jingwen Song, Xiaozhuo Chen. Extracellular ATP induces epithelial-mesenchymal transition: A novel model alternative to TGF-beta for inducing and studying cancer metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1526.

Abstract Metastasis is estimated to be responsible for more than 90% of all cancer-related deaths. Metastasis is initiated by induction of epithelial-mesenchymal transition (EMT) of cancer cells, but factors and mechanisms involved in EMT are far from being fully elucidated. Intratumoral extracellular ATP (eATP), at levels of 100-700 μM or 103 to 104 times higher than in normal tissues, has been known to induce epithelial-mesenchymal-transition (EMT) of cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis. Here we report that eATP, at the concentrations reported in tumors, potently induces expression of matrix metallopeptidases (MMPs), detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-β and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of the macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis and slows down tumor growth in nude mice. Collectively, these results show that eATP functions on these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings which shed light on eATP's initiator and effector roles in almost every step in early metastasis call for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identify eATP and macropinocytosis as novel targets for potentially slowing down EMT, preventing metastasis, and reducing metastasis-related death in cancer patients. Citation Format: Yanyang Cao, Xuan Wang, Maria Evers, Yunsheng Li, Haiyun Zhang, Xiaozhuo Chen. New roles of ATP in metastasis: Extracellular and macropinocytosis internalized ATP work together to induces epithelial to mesenchymal transition and other early steps of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1521.

Abstract Cancer is a major death-causing disease worldwide, and metastasis, a hallmark of cancer, is associated with up to 90% of all cancer-related deaths. Metastasis requires ATP, and the tumor microenvironment (TME) has much higher concentrations of intratumoral and extracellular ATP (eATP) than healthy tissue, implying that eATP may have potential roles in tumor development and progression. Our previous ATP studies, cited by a Nature Review Cancer paper in 20181 showed that eATP is internalized by cancer cell through macropinocytosis, and it promotes cancer cell growth, survival, and drug resistance. However, how intratumoral eATP plays roles in metastasis remains largely unclear. Our 2019 published results show that eATP promotes cancer migration and invasion, initiates epithelial to mesenchymal transition (EMT), alters expression level of EMT related protein markers such as ZEB1, SNAIL, SLUG in human non-small lung cancer cell (NSCLC) lines2. It has been reported that the EMT and cancer stem cells (CSCs) share some molecular links, but the detailed mechanisms involved are still unknown. Recently, we have found the eATP significantly induces the gene expression level of CSC transcription factors (TFs): SOX2, NANOG and OCT4 in A549 cells in a time-dependent manner. We have also found that, after A549 cells were pretreated with 0.5mM for 6 hrs, the ATP-pretreated A549 cells were able to show significantly increased migration and invasion even in the absence of ATP addition. Based on all these results, we hypothesize that extracellular ATP functions as a messenger, a signal facilitator, an energy molecule, and a transcription cofactor to induce EMT and CSC-like features to promote metastasis in NSCLC. RNAseq and metabolomics studies are being used to determine the transcriptional and metabolic changes associated with the ATP induced EMT and CSC process in NSCLC. The completion of the project will not only unravel eATP's roles in EMT and CSC induction and early steps of tumor metastasis, but also potentially provide novel promising therapeutic targets for inhibiting tumor metastasis. Citation Format: Jingwen Song, Maria Evers, Xiaozhuo Chen. Roles of extracellular ATP in inducing EMT and CSC-like changes to promote early steps of metastasis in non-small lung cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1542.

Abstract Background Extracellular ATP (eATP) was shown to induce epithelial–mesenchymal transition (EMT), a very important early process in metastasis, in cancer cells via purinergic receptor signaling. However, the exact induction mechanisms are far from fully known. We previously described that eATP is internalized by cancer cells in vitro and in vivo by macropinocytosis in human non-small cell lung cancer A549 and other cancer cells, drastically elevates intracellular ATP levels, enhances cell proliferation and resistance to anticancer drugs. In this study, we tested the hypothesis that eATP and macropinocytosis-internalized eATP also induces EMT and other early steps of metastasis. Methods Floating cells, fencing, and transwell assays were used to show that ATP induces cell detachment, new colony formation, migration and invasion in human A549 and other lung cancer cells. Western blots were used to detect ATP-induced changes in EMT-related proteins; Confocal microscopy was used to demonstrate ATP-induced metastasis-related cell morphological changes. Inhibitors and siRNA knockdowns were used to determine P2X7’s involvement in the ATP-induced EMT. CRISPR–Cas9 knockout of the SNX5 gene was used to identify macropinocytosis’ roles in EMT and cancer cell growth both in vitro and in vivo. Student t-test and one-way ANOVA were used to determine statistical significance, P < 0.05 was considered significant. Results eATP potently induces expression of matrix metallopeptidases (MMPs), and detachment, EMT, migration, and invasion of lung cancer cells. The induction was independent of TGF-β and semi-independent of P2X7 activation. eATP performs these functions not only extracellularly, but also intracellularly after being macropinocytically internalized to further enhance P2X7-mediated EMT, filopodia formation and other early steps of metastasis. The knockout of macropinocytosis-associated SNX5 gene significantly reduces macropinocytosis, slows down tumor growth, and changes tumor morphology in nude mice. Conclusions Collectively, these results show that eATP's functions in these processes not only from outside of cancer cells but also inside after being macropinocytotically internalized. These findings shed light on eATP’s initiator and effector roles in almost every step in early metastasis, which calls for rethinking and rebalancing energy equations of intracellular biochemical reactions and the Warburg effect, and identifies eATP and macropinocytosis as novel targets for potentially slowing down EMT and preventing metastasis.