Abstract Head and neck squamous cell carcinoma (HNSCC) is a leader in cancer incidence worldwide. Although the introduction of immunotherapy, such as immune checkpoint blockade (ICB), has improved the landscape of cancer treatment in many solid tumors, including HNSCCs, there remain challenges in achieving durable responses with such therapies. Indeed, HNSCC patients often relapse following ICB treatment. While relapse events are high, there is a lack of understanding of the mechanisms underlying cancer immune evasion. These disparities in patient outcomes strongly suggest the existence of genetic variations that underlie their distinct responses to ICB. Although the specific oncogenic mutations responsible for driving relapse in SCC patients remain unclear, it is critical to elucidate the mechanisms of cancer immune evasion in individuals with specific genetic profiles to enhance the precision of immunotherapy. We aim to understand the genetic basis shaping the immune suppressive TME and hypothesize that oncogenic driver mutations play a dominant role in preventing the immune clearance of transformed cells by reprogramming the immune landscape in the tumors. To determine the critical genetic signatures enriched in SCC patients that can impact anti-tumor immunity, we analyzed TCGA data which revealed a strong negative correlation between PIK3CA level and CD8+ T cell signatures. We have identified that activating mutations in the PIK3CA gene, found in 20% of HNSCCs, promote rapid tumor relapse after initial response to anti-PD-L1 and anti-CTLA-4 ICB treatments. Utilizing single-cell analysis, quantitative immune profiling, and multiplexed imaging, our lab showed that tumor-initiating cells (TICs) in SCCs can have an intricate dialogue with myeloid-derived suppressor cells (MDSCs) where TICs secrete factors to enhance MDSC recruitment and suppressive function on cytotoxic T cells. Our lab also identified that SOX2 amplification in PIK3CA mutant SCC could modulate neutrophils and block their interferon responses via activation of fatty acid desaturase 1 (FADS1) to aid neutrophils in maintaining their immune suppressive functions during immunotherapy treatments. As such, this study has led to key mechanistic understanding of how PIK3CA mutation allows cancer cells to shape the immune suppressive responses. Thus, our findings uncover a unique mechanism whereby PIK3CA mutant SCCs critically shape the tumor microenvironment to survive robust immunotherapy and give rise to tumor relapse. Citation Format: Sydney Fisher, Benjamin Nicholson, Weijie Guo, Yuxuan Phoenix Miao. PIK3CA activating mutation reshapes the tumor microenvironment to promote immune evasion in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7792.

Abstract Immunotherapies have transformed cancer treatment, but subsets of patients experience differential responses. Patients with squamous cell carcinomas (SCCs) in various tissues exhibit a high rate of relapse after initial responses to immunotherapies. Recent research highlighted the critical role of a group of stem cell-like tumor-initiating cells (TICs) in driving resistance to anti-tumor immunity and promoting relapse of both cutaneous (CSCCs) and head and neck (HNSCCs) SCCs despite intact antigen presentation. Although TICs drive SCC relapse, they compose less than 5% of the total tumor cell population, so their unique immune resistance mechanisms have been largely overlooked. We analyzed single-cell RNA-seq data profiling the gene signatures of various tumor populations in spontaneous GEMM CSCCs and identified that cytotoxic T lymphocyte-associated protein 2a (Ctla2a) was specifically activated in TICs. Ctla2a is a cysteine peptidase inhibitor with high homology to the I29 inhibitory domain of mouse and human cathepsins. Silencing Ctla2a in SCC cells reduced tumor growth, increased the frequency of granzyme B+ CD8+ T cells infiltrating the tumor, and sensitized SCC tumors to immunotherapy. We have found that the conditioned medium from SCC cells overexpressing Ctla2a is sufficient to blunt granzyme B production in CD8+ T cells and reduce CD8+ T cell antigen-specific cytotoxicity in vitro. Taken together, these results suggest that SCC TICs can secrete Ctla2a to modulate CD8+ T cell anti-tumor cytotoxicity. This interaction may serve as a target for next-generation immunotherapy that is able to blunt TIC-specific immune resistance. Citation Format: Benjamin Nicholson, Sydney Fisher, Matthew Wren, Weijie Guo, Yuxuan Phoenix Miao. Identification of a secretory immune regulatory ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6990.

Although immunotherapy has been a key innovation in cancer treatment, tumor relapse still poses a major challenge for many patients. Recent research has shown that cancer relapse can be caused by a special group of cancer cells enriched with stemness signatures that survive treatments and repopulate tumors. These cells are referred to as tumor-initiating stem cells (tSCs). The mechanisms by which tSCs survive immunotherapy to drive cancer relapse are still unclear. Using a spontaneous squamous cell carcinoma (SCC) mouse model, we found that tSCs have elevated expression of CD80, a surface molecule that provides co-stimulation to T cells. Notably, the loss of CD80 expression on SCC cells led to diminished tumor growth by blunting the expansion of intratumoral regulatory T cells (Tregs). This interaction between epithelial stem cell and Treg cells can also be found during wound repair where CD80 expression by hair follicle stem cells promotes expansion of Tregs in the wound bed. Interestingly, we found that CD80 on epithelial stem cells facilitates Treg expansion by an unconventional mechanism in which the Foxp3 can be directly induced in preactivated tumor infiltrating CD4+ T cells. Collectively, this study reveals a unique mechanism by which tSCs may sculpt an immunosuppressive niche to evade killing and facilitate tumor relapse. Jennifer L. Good, Weijie Guo, Jingyun Luan, Benjamin T. Nicholson, Yuxuan Miao. Tumor-initiating stem cells utilize the immunomodulatory ligand CD80 to promote Treg expansion and cancer relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 927.

The heterogeneous nature of tumor-associated neutrophils (TANs) has been recognized, but how different cell states of TANs emerge, evolve, distribute, and impact cancer immunotherapy efficacy remain elusive. Using single-cell RNA sequencing, spatial transcriptomics, and genetic manipulations, we show that anti-PDL1 + CD40 agonist immunotherapy can induce interferon responses in TANs, allowing them to regain anti-tumor activities in squamous cell carcinomas (SCCs). In contrast, TANs residing at the tumor-stroma interface can preserve their immune-suppressive state. Importantly, we identify a group of SOX2High tumor-initiating stem cells (tSCs) at the tumor-stroma interface that upregulate fatty acid desaturase 1 (Fads1) to produce arachidonic acid (AA). This tSC-specific pathway enhances the prostaglandin E2 (PGE2) signaling in TANs, which can disrupt the interferon response and prevent the interferon-induced anti-tumor functions in TANs. By fine-tuning the plasticity of neutrophils, tSCs shape neutrophil heterogeneity and sculpt a protective micro-niche to survive from immunotherapy and drive cancer relapse.

Immunotherapies have transformed cancer treatment, but subsets of patients experience differential responses. Patients with squamous cell carcinomas (SCCs) in various tissues exhibit a high rate of relapse after their initial response to immunotherapies. Recent research highlighted the critical roles of a group of stem cell-like tumor-initiating cells (TICs) in driving the relapse of both cutaneous (CSCCs) and head and neck (HNSCCs). These TICs exhibit superior resistance to anti-tumor immunity despite their intact antigen presentation. Although TICs are the root cause of SCC relapse, they compose less than 5% of the total tumor cell population. Therefore, their unique immune resistance mechanisms have been largely overlooked. Here, we aim to identify the special immune regulatory mechanisms that are specifically activated in TICs endowing them with stronger immune resistance. By analyzing single-cell RNA-seq data profiling the gene signatures of various tumor populations in CSCCs, we identified cytotoxic T lymphocyte antigen 2a (Ctla2a) was specifically activated in TICs. Ctla2a is characterized as a cysteine peptidase inhibitor and has high homology to the I29 inhibitory domain in the proregion of mouse and human cathepsins. Silencing Ctla2a in SCC cells reduced tumor growth, enhanced CD8+ T cell infiltration, and elevated their antigen-specific cytotoxicity. Importantly, we have found that the conditioned medium collected from SCC cells overexpressing Ctla2a is sufficient to blunt the cytokine production of CD8 T cells, suggesting that Ctla2a proteins are secreted from TICs to modulate T cell immunity. Ongoing aims focus on 1) how the peptidase inhibitor activity of Ctla2a reduces cytotoxic cytokine production in CD8+ T cells and 2) whether the I29 inhibitory domain of human cathepsins is capable of conferring the immune-suppressive activity observed in Ctla2a expressing SCCs. Taken together, these results suggest that TICs can secrete these cathepsin inhibitory peptides to perform moonlighting functions and modulate CD8+ T cell anti-tumor cytotoxicity. This interaction may serve as a target for next-generation immunotherapy that is able to blunt TIC-specific immune resistance. Benjamin T. Nicholson, Ama Ofori, Jennifer Good, Weijie Guo, Yuxuan Miao. Identification of a stem cell-specific secretory immune regulatory ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 907.

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

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.

Approximately 20% of patients with myeloproliferative neoplasms (MPNs) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type 1 or type 2. While type 2 CALR mutant proteins retain many of the Ca2+ binding sites present in the wild type protein, type 1 CALR mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain yet unexplored. Here, we show that the loss of Ca2+ binding residues in the type 1 mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1a/XBP1 pathway of the unfolded protein response. Genetic or pharmacological inhibition of IRE1a/XBP1 signaling induces cell death only in type 1 mutant but not type 2 mutant or wild type CALR-expressing cells, and abrogates type 1 mutant CALR-driven MPN disease progression in vivo.

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.