Identification of MUC1-c as a target for the treatment of squamous cell carcinomas

Abstract

The present disclosure relates to a novel method of treating squamous cell carcinoma (SCC), and in particular head and neck squamous cell carcinoma (HSSCC) and cutaneous carcinoma (CC) using MUC1-C inhibitors. Also described herein is a novel method of suppressing the progression of premalignant lesions to a squamous cell carcinoma.

Description

BT Ref.91016- 420148 DFCI3434.WO01WO IDENTIFICATION OF MUC1-C AS A TARGET FOR THE TREATMENT OF SQUAMOUS CELL CARCINOMAS RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 336,621, filed on April 12, 2024, which is incorporated by referenced herein in its entirety. STATEMENT REGARDING SEQUENCE LISTING

[0002] The sequence listing associated with this application is provided in XML format in lieu of paper copy, and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is “91016-408411_SeqListing.xml”. The XML file is 89.6 KB, and was created on April 12, 2024, and is being submitted electronically, concurrent with the filing of this application. FIELD

[0003] The present disclosure relates to a novel method of treating squamous cell carcinoma (SCC), and in particular head and neck squamous cell carcinoma (HSSCC) and cutaneous carcinoma (CC) using MUC1-C inhibitors. Also described herein is a novel method of suppressing the progression of premalignant lesions to a squamous cell carcinoma. BACKGROUND

[0004] The MUC1-C protein has been found to be aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression to cancer, including lung, breast, pancreatic and ovarian cancer. (See, Chen et al., 2021). Studies of HNSCC cell lines demonstrate that MUC1-C integrates chronic activation of the STAT1 inflammatory pathway with induction of the ΔNp63 and SOX2 genes that are aberrantly expressed in HNSCCs, and facilitates NOTCH3 expression, self-renewal capacity and tumorigenicity. The MUC1 gene has been found to be upregulated in advanced head and neck SCCs (HNSCCs); however, there is no known association of MUC1-C in the progression of squamous cell carcinomas.BT Ref.91016- 420148 DFCI3434.WO01WO

[0005] Squamous cell carcinoma (SCC) includes cancers arising on the epidermis as well as mucous membrane surfaces, such as skin as well as head and neck cancers. The past 30 years have seen the rate of SCC rise by approximately 200%. Current treatments for SCC include surgery, radiation, chemotherapy, and immunotherapy, but surgery remains the most effective treatment with the least side effects. However, in certain cases, SCC can become recurrent or progressive, even after surgery, for example, where microscopic remnants of the SCC are left behind to proliferate. Recurrent or untreated SCC can lead to a more progressive form, which can have a poor prognosis. Thus, more effective treatments for SCC and to prevent or treat progressive SCC are desirable. SUMMARY

[0006] One embodiment described herein is a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising administering a therapeutically effective amount of a MUC1-C inhibitor.

[0007] In one aspect, the squamous cell carcinoma is a head and neck SCC or a cutaneous SCC. In another aspect, the squamous cell carcinoma is localized to a tissue. In one aspect, MUC1-C inhibitor is a peptide, a small molecule, a biologic, or a nucleic acid. In one aspect, the inhibitor is a GO-203 peptide. In another aspect, inhibitor is salinomycin. In another aspect, the inhibitor is an anti-sense RNA oligonucleotide. In one aspect, the inhibitor is formulated as a topical or an injectable. In one aspect, the administration is localized. In another aspect, the administration is prior to a surgical excision of the SCC.

[0008] In one aspect, the method further comprises administration of one or more of a chemotherapy, immunotherapy, or a surgical excision of the SCC. In another aspect, the chemotherapy comprises one or more of a cisplatin or 5-fluorouracil. In another aspect, the immunotherapy comprises one or more of cetuximab, pembrolizumab or nivolumab.

[0009] Another embodiment described herein is a method of suppressing chronic inflammation in a patient in need thereof comprising administering a therapeutically effective amount of a MUC1-C inhibitor.BT Ref.91016- 420148 DFCI3434.WO01WO

[0010] Another embodiment described herein is a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising administering a therapeutically effective amount of a composition comprising means for inhibiting MUC1-C expression or activity and a pharmaceutically acceptable excipient.

[0011] Yet another embodiment is a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising: (a) administering a first line therapeutic composition comprising a MUC1-C inhibitor; (b) performing a second line therapy comprising a surgery; wherein the first line therapy is administered local to the premalignant lesion; and wherein the second line therapy comprises an excision surgery local to the premalignant lesion.

[0012] In one aspect, the squamous cell carcinoma is a head and neck squamous cell carcinoma or a cutaneous squamous cell carcinoma. In another aspect, the first line therapeutic composition is administered according to a dosing regimen.

[0013] Another embodiment described herein is a method of treating recurrent or progressive squamous cell carcinoma, comprising administering to a patient in need thereof an effective amount of (a) a MUC1-C inhibitor disclosed herein, or (b) a pharmaceutical composition thereof. In one aspect, the the pharmaceutical composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

[0014] Another embodiment described herein is a use of (a) a MUC1-C inhibitor disclosed herein, or (b) a pharmaceutical composition thereof for treating recurrent or progressive squamous cell carcinoma. In one aspect, the pharmaceutical composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

[0015] Another embodiment described herein is a MUC1-C inhibitor disclosed herein or pharmaceutical composition thereof for use in therapy. In one aspect the pharmaceutical composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.BT Ref.91016- 420148 DFCI3434.WO01WO

[0016] Another embodiment described herein is a MUC1-C inhibitor disclosed herein or pharmaceutical composition thereof for use in treating recurrent or progressive squamous cell carcinoma. In one aspect, the composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion. BRIEF DESCRIPTION OF THE FIGURES

[0017] Figure 1 is an embodiment showing expression of MUC1 in HNSCC tumors and effects of silencing MUC1-C on HNSCC cell clonogenicity. Figure 1A depicts analysis of primary (P) and metastatic (M) HNSCC tissues for MUC1 expression using the GSE136037 dataset. Figure 1B describes how CAL27 / CshRNA and MUC1shRNA cells were analyzed for MUC1-C mRNA levels (left). The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). Figure 1C describes stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for CshRNA cells (assigned a value of 1)(right). Figure 1D describes the amino acid sequence of the MUC1-C cytoplasmic domain (CD) highlighting direct interactions with JAK1 and STAT1. Figures E and F describe how CAL27 cells expressing the indicated vectors were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins (E). Cells were analyzed for colony formation (F). Shown are representative photomicrographs of stained colonies (upper). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for control cells (assigned a value of 1)(lower).

[0018] Figure 2 is an embodiment showing effects of silencing MUC1-C on HNSCC cell clonogenic survival. Figure 2A depicts a Volcano plot of down- and up-regulated genes in metastatic vs primary HNSCCs in the GSE136037 dataset (left). Levels of MUC1 expression in primary and metastatic HNSCCs (right). Figure 2B describes CAL27 / tet- CshRNA cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C mRNA levels (left). The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left).BT Ref.91016- 420148 DFCI3434.WO01WO Lysates were immunoblotted with antibodies against the indicated proteins (right). C. CAL27 / tet-CshRNA cells treated with vehicle or DOX for 7 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1)(right).

[0019] Figure 3 is an embodiment showing how MUC1-C regulates a global transcriptional program enriched for STAT / IRF signaling in HNSCC cells. Figure 3A describes how RNA-seq was performed on CAL27 / tet-MUC1shRNA and HSC3 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days. Volcano plots depicting downregulated (blue) and upregulated (red) DEGs (FDR<0.05; FC>2). Highlighted are the top DEGs by significance and magnitude. Figure 3B depicts common down-regulated and up-regulated genes in CAL27 and HSC3 cells with MUC1-C silencing. Figure 3C describes purified chromatin from CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was immunoblotted with antibodies against the indicated proteins. D and E. Candidate enrichment plots for the HALLMARK INTERFERON ALPHA RESPONSE (C), HALLMARK INTERFERON GAMMA RESPONSE (D) gene signatures. Figures 3F and 3G describe box plots of selected ISG expression in CAL27 (F) and HSC3 (G) cells with MUC1-C silencing.

[0020] Figure 4 is an embodiment showing MUC1-C regulates the type I and IFN pathways in CAL27 and HSC3 cells. Figures 4A and 4B describe HSC3 / tet-CshRNA (A) and HSC3 / tet-MUC1shRNA (B) cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C mRNA levels (left). The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). Figures 4C and 4D describe FaDu / tet-CshRNA (C) and FaDu / tet-MUC1shRNA (D) cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C mRNA levels (left). The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). Figures 4E and 4F describe HSC3 / tet-MUC1shRNA (E) and FaDu / tet- MUC1shRNA (F) cells treated with vehicle or DOX for 7 days were analyzed wereBT Ref.91016- 420148 DFCI3434.WO01WO analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle treated cells (assigned a value of 1)(right). Figure 4G describes the effects of silencing MUC1-C on the indicated HALLMARK gene signatures.

[0021] Figure 5 is an embodiment showing MUC1-C regulating expression of PRRs and effectors of the type I and II IFN pathways. Figure 5A describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 6 days were analyzed for RIG-I and MDA5 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 5B describes CAL27 cells expressing the indicated vectors were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. Figure 5C describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for STAT1, STAT2 and IRF9 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle- treated cells (assigned a value of 1). Figure 5D describes CAL27 cells expressing the indicated vectors were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. Figure 5E describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for OAS1, MX1 and ISG15 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 5F describes CAL27 cells expressing the indicated vectors were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. Figure 5G describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for GBP-1, IDO-1 and WARS mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 5H depicts CAL27 cells expressing the indicated vectors were treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins.

[0022] Figure 6 is an embodiment showing effects of targeting MUC1-C on effectors of the type I and II IFN pathways. Figure 6A – Figure 6D. Lysates from HSC3 / tet-BT Ref.91016- 420148 DFCI3434.WO01WO MUC1shRNA cells treated with vehicle of DOX for 7 days were immunoblotted with antibodies against the indicated proteins.

[0023] Figure 7 is an embodiment showing MUC1-C / STAT1 signaling regulates ΔNp63 expression. Figure 7A describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days analyzed for ΔNp63 gene transcription (left) and mRNA (right) levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 7B describes CAL27 / CshRNA and CAL27 / STAT1shRNA cells analyzed for ΔNp63 gene transcription (left) and mRNA (right) levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 7C describes schema of the ΔNp63 gene with highlighting localization of the PLS region that contains potential STAT1 binding motifs. Figure 7D describes soluble chromatin from CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-MUC1-C and anti-STAT1. The DNA samples were amplified by qPCR with primers for the ΔNp63 PLS region. The results (mean±SD of 3 determinations) are expressed as percentage of the input DNA for each sample. Figure 7E depicts CAL27 cells expressing the indicated vectors treated with vehicle or DOX for 7 days. Lysates were immunoblotted with antibodies against the indicated proteins. Figure 7F describes lysates from CAL27 / CshRNA and CAL27 / STAT1shRNA cells were immunoblotted with antibodies against the indicated proteins.

[0024] Figure 8 is an embodiment showing regulation of ΔNp63 expression in HSC3 cells. Figure 8A describes HSC3 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and analyzed for ΔNp63 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 8B describes HSC3 / CshRNA and CAL27 / STAT1shRNA cells analyzed for ΔNp63 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 8C describes lysates from HSC3 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. Figure 8D describes lysates from HSC3 / CshRNA and CAL27 / STAT1shRNA cells immunoblotted with antibodies against the indicated proteins.BT Ref.91016- 420148 DFCI3434.WO01WO

[0025] Figure 9 is an embodiment showing MUC1-C / STAT1 signaling regulates activation of the SOX2 gene. Figure 9A depicts CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days analyzed for SOX2 gene transcription (left) and mRNA (right) levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells(assigned a value of 1). Figure 9B describes CAL27 / CshRNA and CAL27 / STAT1shRNA cells analyzed for SOX2 gene transcription (left) and mRNA (right) levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Figure 9C describes schema of the SOX2 gene with highlighting localization of the PLS region that contains STAT1 binding motifs. Figure 9D describes soluble chromatin from CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with anti-MUC1-C and anti-STAT1. The DNA samples were amplified by qPCR with primers for the SOX2 PLS region. The results (mean±SD of 3 determinations) are expressed as percentage of input DNA for each sample. Figure 9E describes lysates from CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days immunoblotted with antibodies against the indicated proteins. Figure 9F describes lysates from CAL27 / CshRNA and CAL27 / STAT1shRNA cells immunoblotted with antibodies against the indicated proteins.

[0026] Figure 10 is an embodiment showing regulation of SOX2 expression in HSC3 cells. Figure 10A describes HSC3 / tet-MUC1shRNA cells treated with vehicle or DOX for 10 days and analyzed for SOX2 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle- treated cells (assigned a value of 1). Figure 10B describes HSC3 / CshRNA and HSC3 / STAT1shRNA cells analyzed for SOX2 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells(assigned a value of 1).

[0027] Figure 11 is an embodiment showing MUC1-C regulates NOTCH3 expression and HNSCC cell self-renewal capacity. Figure 11A describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days analyzed for NOTCH3 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1)(left). Lysates wereBT Ref.91016- 420148 DFCI3434.WO01WO immunoblotted with antibodies against the indicated proteins (right). Figure 11B depicts GSEA of RNA-seq data from CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days using the REACTOME SIGNALING BY NOTCH3 gene signature. Figure 11C indicated CAL27 cells treated with vehicle or DOX for 7 days were analyzed for tumorsphere formation. Shown are representative images of the tumorspheres (bar represents 100 microns). Sphere forming efficiency (SFE) is expressed as the mean±SD of three independent replicates relative to that obtained for vehicle treated cells (assigned a value of 1). Figure 11D depicts lysates from CAL27 cells treated with vehicle or 5 μM GO-203 for 3 days were immunoblotted with antibodies against the indicated proteins. Figure 11E depicts CAL27 cells were treated with vehicle or 5 μM GO-203 while they were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle treated cells (assigned a value of 1)(right). Figure 11F depicts CAL27 cells were treated with vehicle or 5 μM GO-203 while they were analyzed for tumorsphere formation. Shown are representative images of the tumorspheres (bar represents 100 microns). Sphere forming efficiency (SFE) is expressed as the mean±SD of three independent replicates relative to that obtained for untreated cells (assigned a value of 1). Figures 11G and 11H depict six-week old nude mice were injected subcutaneously in the flank with 1 × 107CAL27 cells. Mice pair- matched into groups of five mice each when tumors reached 100–150 mm3were treated with vehicle control or GO-203 for the indicated days. Tumor volumes are expressed as the mean±SEM for five mice (G). Lysates from control and GO-203-treated tumors were immunoblotted with antibodies against the indicated proteins.

[0028] Figure 12 is an embodiment showing effects of targeting MUC1-C with the GO3- 203 inhibitor. Figure 12A describes CAL27 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and analyzed for NOTCH3 mRNA levels. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle- treated cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). Figure 12B describes GSEA of RNA-seq data from HSC3 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days using the REACTOME SIGNALING BY NOTCH3 gene signature. Figure 12C depicts the indicatedBT Ref.91016- 420148 DFCI3434.WO01WO HSC3 cells treated with vehicle or DOX for 7 days and analyzed for tumorsphere formation. Shown are representative images of the tumorspheres (bar represents 100 microns). Sphere forming efficiency (SFE) is expressed as the mean±SD of three independent replicates relative to that obtained for vehicle treated cells (assigned a value of 1). Figure 12D describes lysates from HSC3 cells treated with vehicle or 5 μM GO-203 for 2 days were immunoblotted with antibodies against the indicated proteins. Figure 12E describes HSC3 cells treated with vehicle or 5 μM GO-203 analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle treated cells (assigned a value of 1)(right). Figure 12F describes HSC3 cells treated with vehicle or 5 μM GO-203 and analyzed for tumorsphere formation. Shown are representative images of the tumorspheres (bar represents 100 microns). Sphere forming efficiency (SFE) is expressed as the mean±SD of three independent replicates relative to that obtained for untreated cells (assigned a value of 1).

[0029] Figure 13 is an embodiment showing single-cell profiling of the expression of MUC1 and related genes in HNSCC. Figures 13A to 13C depict a UMAP representation of total cells analyzed from the GSE118389 dataset of HPV-negative / HPV-positive HNSCC samples (Figure 13A), GSE118389 dataset of HPV-negative samples (Figure 13B), and GSE118389 dataset of HPV-positive samples (Figure 13C) (left panels). Distributions of MUC1 expression in individual cells (middle panels). MUC1 expression in individual cells in each cell type (right panels). Figure 13D is a model depicting the present findings that MUC1-C integrates activation of STAT1, IFN type I / II signaling and ISG expression with regulation of ΔNp63, SOX2 and NOTCH3 in driving the HNSCC CSC state that promotes DNA damage resistance and immune evasion.

[0030] Figure 14 is an embodiment showing single-cell profiling of the expression of MUC1 and related genes in HNSCC. Figure 14A. Expression of STAT1, TP63, SOX2 and NOTCH3 across UMAP projections (top) and in each cell type (below) in HPV- negative / HPV-positive HNSCC samples. Figures 14B and 14C depicts a correlation analysis between MUC1 and STAT1, TP63, SOX2, NOTCH3 expression in identified malignant cells. Figure 14D depicts expression of STAT1, TP63, SOX2 and NOTCH3 across UMAP projections (top) and in each cell type (below).BT Ref.91016- 420148 DFCI3434.WO01WO

[0031] Figure 15 is an embodiment showing silencing MUC1-C in cutaneous SCC13 cells downregulates ΔNp63, NOTCH1 / 3 and HES1 mRNA levels. Human SCC13 cutaneous SCC cells expressing tet-CshRNA (tet1) or tet-MUC1shRNA (tet8) were treated with vehicle or DOX for 5 days and analyzed for indicated transcripts. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1).

[0032] Figure 16 is an embodiment showing silencing MUC1-C in SCC13 cells suppresses ΔNp63 and NOTCH1 protein levels. Lysates from SCC13 cells expressing tet-CshRNA (tet1) or tet-MUC1shRNA (tet8) treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins.

[0033] Figure 17 is an embodiment showing targeting MUC1-C in SCC13 cells with the GO-203 inhibitor suppresses ΔNp63 and NOTCH1 protein levels. Lysates from SCC13 cells treated with vehicle or 5 μM GO-203 for 2 days were immunoblotted with antibodies against the indicated proteins.

[0034] Figure 18 is an embodiment showing targeting MUC1-C in SCC13 cells with the GO-203 inhibitor suppresses self-renewal capacity. SCC13 cells treated with vehicle or 5 μM GO-203 for 2 days were analyzed for tumorsphere formation. Shown are representative images of the tumorspheres. The number of tumorspheres is expressed as the mean±SD of three independent replicates. DETAILED DESCRIPTION

[0035] The MUC1 gene evolved in mammals to protect barrier tissues from loss of homeostasis by inflammatory insults (See, Kufe et al., 2020, Cancers (Basel) 2022 Cancer Metastasis Treat 2022). The MUC1-C subunit activates EMT, epigenetic reprogramming and repair responses to stress that, if prolonged as in settings of chronic inflammation, contribute to cancer progression. MUC1-C has been largely associated with adenocarcinomas that arise from barrier glandular epithelial cells lining internal organs. It has been found that MUC1 is upregulated in metastatic HNSCCs that are derived from barrier stratified squamous epithelial cells (See, Dotto et al., 2018). Advanced HNSCCs invariably become refractory to multi-modality treatment with chemotherapy, radiotherapy and immunotherapy in association with development ofBT Ref.91016- 420148 DFCI3434.WO01WO DNA damage resistance and immune evasion (See, Rao et al., 2023, Chakrobarty et al., 2023, and Papalouka et al., 2023).

[0036] Furthermore, chronic inflammation is an established driver of cancer progression and therapeutic resistance, albeit by unifying mechanisms that have remained unclear (See, Greten et al., 2019). MUC1-C is activated in barrier tissues in response to inflammation (See, Kufe et al., Cancers (Basel) 2022). Squamous epithelia of the head and neck are exposed to cigarette smoke and alcohol that contribute to chronic inflammation (See, Dotto et al., 2016). In settings of chronic inflammation with repetitive cycles of damage and repair, prolonged MUC1-C activation becomes established in promoting cancer progression (See, Kufe et al., Cancers (Basel) 2022).

[0037] The present disclosure describes a novel method of treating squamous cell carcinoma, and in particular head and neck squamous cell carcinoma and cutaneous carcinoma using a MUC1-C inhibitor, such as progressive or recurrent head and neck squamous cell carcinoma and cutaneous carcinoma.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0039] As used herein, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.

[0040] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In some embodiments, the terms "about" or "approximately" when preceding a numerical value indicates the value plus or minus a range of 10%, 5%, or 1%.BT Ref.91016- 420148 DFCI3434.WO01WO

[0041] As used herein, "an effective amount" refers to an amount that causes relief of symptoms of a disorder or disease as noted through clinical testing and evaluation, patient observation, and / or the like. An "effective amount" may further designate a dose that causes a detectable change in biological or chemical activity. The detectable changes may be detected and / or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" may designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and / or promotes improvement in the condition of interest. An "effective amount" may further refer to a “therapeutically effective amount”.

[0042] As used herein, the term “antisense oligonucleotide” means a plurality of linked nucleosides, at least a portion of which, is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity. In one aspect described herein, oligonucleotides comprise one or more of deoxyribonucleosides (DNA) and / or ribonucleosides (RNA). In one aspect, the antisenense oligonucleotide is complementary to the nucleic acid encoding MUC1-C. Exemplary antisense oligonucleotides include siRNA, shRNA and the like.

[0043] Antibodies have long been used to direct pharmaceuticals to cells by specifically targeting cell surface receptors. The present disclosure thus contemplates the use of appropriate antibodies alone or as an antibody-drug conjugate in conjunction with any appropriate therapeutic agent. In one aspect described herein, an anti-MUC1-C antibody may be a MUC1-C inhibitor. In another aspect, the antibodies 3D1 and 7B8 (against the MUC1-C extracellular domain) are MUC1-C inhibitors and may be conjugated to any of appropriate therapeutic agent. However, any suitable antibody may be used.

[0044] As used herein, the term "individual" and "subject" are often used interchangeably and refer to any human or domestic animal that may be treated with the methods disclosed herein. Suitable subjects (e.g., patients) include humans and domestic animals or pets (such as a cat or dog). Non–human primates and human patients are included. In one embodiment, subjects may include human patients that have been diagnosed with squamous cell carcinoma, including but not limited to: head and neck squamous cell carcinoma (HNSCC), skin or cutaneous squamous cellBT Ref.91016- 420148 DFCI3434.WO01WO carcinoma (CSCC), adenoid squamous cell carcinoma, intra-epidermal squamous cell carcinoma, large cell keratinizing squamous cell carcinoma, lympho-epithelial carcinoma, papillary squamous cell carcinoma, or spindle cell carcinoma. As used herein, the term "patient" refers to a subject that may receive a treatment of a disease or condition.

[0045] As used herein, the term “MUC1-C inhibitors” refers to any molecule that inhibits or disrupts expression of MUC1-C nucleic acid or inhibits or disrupts MUC1-C oncoprotein activity.

[0046] As used herein, the term “neoadjuvant therapy” refers to a treatment given as a first step to shrink a tumor before the main treatment, which is primarily a surgical treatment, for example, Mohs surgery to remove a skin cancer lesion.

[0047] As used herein, the term “premalignant lesion” or “premalignancy” refers to an area of tissue having abnormal cells with an increased risk of developing into cancer.

[0048] As used herein, "treatment", "treat", and "treating" refer to reversing, alleviating, mitigating, or slowing the progression of, or inhibiting the progress of, a disorder or disease or symptoms associated with such disorder or disease, and as described in more detail herein.

[0049] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0050] Described herein is a method of treating squamous cell carcinoma, a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma, and a method of suppressing chronic inflammation, in a patient in need thereof, all of which comprises administration of a MUC1-C inhibitor.

[0051] Squamous cell carcinomas are commonly found in the head and neck region, and on the skin. Rarely metastatic, these malignancies are localized and may advance and invade surrounding structures. (See, Nuttall et al., 2023). Symptoms may include skin changes such as raised growths, scaly skin, open sores, or brown spots. Current treatments include surgical excision and / or radiotherapy. The goal of surgical excisionBT Ref.91016- 420148 DFCI3434.WO01WO is to obtain complete tumor resection which the lowers risk of complication or recurrence. (See, Moreno-Ramirez et al., 2021). Neoadjuvant therapies may be administered prior to surgery may support excisions. Where SCC is advanced, and surgical excision would result in major anatomic mutilation or disfiguration, alternative therapies include radiation therapy and / or systemic therapy.

[0052] With the role of MUC1-C in chronic inflammation and / or progression of premalignant lesions to an SCC, inhibitors targeting MUC1-C may also be used to treat these cancers. The MUC1-C gene is 477 nucleotides in length and when expressed, is a transmembrane protein with a 58 amino acid long extracellular domain, a 28 amino acid long transmembrane domain and a 72 amino acid long cytoplasmic domain (See, Kufe, 2009). Without being bound by any theory, the cytoplasmic domain is believed to be involved in nuclear import and activation of various inflammatory pathways. (See Kufe, 2009). SEQ ID NO: 60 describes the DNA sequence of MUC1-C and SEQ ID NO: 61 describes the amino acid sequence of MUC1-C with the extracellular domain italicized, the transmembrane domain underlined, and the cytoplasmic domain in lower caps.

[0053] SEQ ID NO: 60: DNA sequence of MUC1-C

[0054] TCTGTGGTGGTACAATTGACTCTGGCCTTCCGAGAAGGTACCATCAATGTCC ACGACGTGGAGACACAGTTCAATCAGTATAAAACGGAAGCAGCCTCTCGATATAAC CTGACGATCTCAGACGTCAGCGTGAGTGATGTGCCATTTCCTTTCTCTGCCCAGTC TGGGGCTGGGGTGCCAGGCTGGGGCATCGCGCTGCTGGTGCTGGTCTGTGTTCTacgggcagctggacatctttccagcccgggatacctaccatcctatgagcgagtaccccacctaccacacccatgggcg ctatgtgccccctagcagtaccgatcgtagcccctatgagaaggtttctgcaggtaatggtggcagcagcctctcttacaca aacccagcagtggcagccacttctgccaacttgtag (SEQ ID NO: 60).

[0055] SEQ ID NO: 61: Amino acid sequence of MUC1

[0056] SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQ SGAGVPGWGIALLVLVCVLVALAIVYLIALAVcqcrrknygqldifpardtyhpmseyptyhthgryvpps stdrspyekvsagnggsslsytnpavaatsanl* (SEQ ID NO: 61).

[0057] Thus any molecules acting as inhibitors of MUC1-C may provide a therapeutic benefit and are contemplated herein. Exemplary molecules include small moleculeBT Ref.91016- 420148 DFCI3434.WO01WO therapies, such as salinomycin. In embodiments, the MUC1-C inhibitor is salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof. Salinomycin is traditionally an antibacterial and that functions as an ionophore to facilitate transport of cations, however it is has been found be an anti-cancer agent for use with breast cancer, leukemia, lymphoma, and colon cancer. Salinomycin has also been found to be potent inhibitor of MUC1-C oncoprotein (See, Kufe et al., 2024).

[0058] In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) is salinomycin, as follows:Salinomycin (2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5- ethyl-5-hydroxy-6-methyloxan-2-yl]-15-hydroxy-3,10,12-trimethyl-4,6,8- trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5- methyloxan-2-yl]butanoic acid In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) is a salinomycin derivative. In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) is ironomycin, as follows:BT Ref.91016- 420148 DFCI3434.WO01WO H3C CH3OH O CH O33(2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5- ethyl-5-hydroxy-6-methyloxan-2-yl]-3,10,12-trimethyl-15-(prop-2-ynylamino)-4,6,8- trioxadispiro[4.1.57.35]17entadic-13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5- methyloxan-2-yl]butanoic acid

[0059] Peptide inhibitors include GO-203, a D-amino acid peptide that blocks MUC1-C homodimerization and is effective in inhibiting growth and survival of MUC1-positive colorectal cancer cells in mouse xenograft models. In embodiments, the MUC1-C inhibitor is GO-203, a derivative thereof, or a pharmaceutically acceptable salt thereof. In embodiments, GO-203 is a D-amino acid sequence comprising CQCRRKN (SEQ ID NO: 62) and a poly-R domain covering the amino-terminal cysteine. In embodiments, the poly-R domain is (R)2-9. In embodiments, the GO-203 peptide is less than 20 amino acids. Additional GO-203 derivatives that are useful in embodiments of the present disclosure can be found in US Pat. Pub Nos.2010 / 0125055, 2012 / 0172312, 20120045502, 2014 / 0322332, 2015 / 0152152, each of which is incorporated by reference in its entirety on April 12, 2024. It is also effective in xenografts models of breast, prostate, lung and certain hematologic malignancy (See, Ahmad et al., 2017).

[0060] Also contemplated herein are anti-MUC1-C antibodies, such as the human monoclonal antibody 3D1 (See, Kufe et al., 2022). Antibodies of use herein may be further conjugated to appropriate MUC1-C inhibitors (such as salinomycin or ironomycin), in the form of antibody-drug conjugates.

[0061] In embodiments, the MUC1-C inhibitor is a MUC1-C antibody, fragment thereof, antibody-drug conjugate thereof, or pharmaceutical composition thereof. In embodiments, the antibody or fragment thereof binds MUC-1C. In embodiments, theBT Ref.91016- 420148 DFCI3434.WO01WO MUC1-C inhibitor is a pharmaceutical composition, comprising a (i) MUC1-C antibody, fragment thereof, or antibody-drug conjugate thereof, and (ii) one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0062] In embodiments, the antibody or fragment thereof binds SEQ ID NO: 57 to MUC- 1C extracellular domain (ECD): SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAG (SEQ ID NO: 57, MUC1-C / ECD)

[0063] In embodiments, the antibody or fragment thereof binds the MUC1-C / ECD at the α3 helix (VHDVETQFNQY, SEQ ID NO: 58) or α4 helix (TEAASPYRN, SEQ ID NO: 59).

[0064] In embodiments, the antibody or fragment thereof comprises a variable region and a constant region, wherein the variable region comprises framework regions and a complementary determining means for binding MUC1-C (SEQ ID NO: 61) or MUC-1C ECD (SEQ ID NO: 57). In embodiments, the complementary determining means for binding comprises binding SEQ ID NO: 57, 58, 59, or 61. In embodiments, the constant region is IgG or IgM. In embodiments, the constant region is IgG. In embodiments, the constant region is IgG1. In embodiments, the constant region is IgG2. In embodiments, the constant region is IgG2a.

[0065] In embodiments, the antibody or fragment thereof comprises heavy chain (HC) variable region (HCVR) and a light chain (LC) variable region (LCVR). In embodiments, the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3. In embodiments, the LCVR further comprises CDRs LCDR1, LCDR2, and LCDR3. Table 1 includes example sequences that can be used in the present disclosure.BT Ref.91016- 420148 DFCI3434.WO01WO Table 1. Example sequences for use in the present disclosure. Clone HCVR LCVR CDR1 CDR2 CDR3V O: L O: L O: L O: O:BT Ref.91016- 420148 DFCI3434.WO01WO LQKPGQSPKLLIYKVSNRFSG SEQ ID NO: SEQ ID NO: SEQ ID NO: VPDRFSGSGSGTDFTLKINRV 30 31 32 O: O: O: P O: O:BT Ref.91016- 420148 DFCI3434.WO01WO LCVR RASQSIGTS YASESIS QQSNNWP DILLTQSPAILSVSPGERVSFS IH LT O: O: Y O:

[0066] In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 1, and the amino acid sequence of the LCVR is SEQ ID NO: 2. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 3, and the amino acid sequence of the LCVR is SEQ ID NO: 4. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 5, and the amino acid sequence of the LCVR is SEQ ID NO: 6. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 7, and the amino acid sequence of the LCVR is SEQ ID NO: 8. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 9, and the amino acid sequence of the LCVR is SEQ ID NO: 10. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 11, and the amino acid sequence of the LCVR is SEQ ID NO: 12. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 13, and the amino acid sequence of the LCVR is SEQ ID NO: 14.

[0067] In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acidBT Ref.91016- 420148 DFCI3434.WO01WO sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0068] In embodiments, the antibody or fragment thereof comprises an antibody fragment. In embodiments, antibody fragment comprises a F(ab). In embodiments, the F(ab) comprises an HCVR and a LCVR. In embodiments, the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3. In embodiments, the LCVR comprises LCDRs LCDR1, LCDR2, and LCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ IDBT Ref.91016- 420148 DFCI3434.WO01WO NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0069] In embodiments, the antibody fragment comprises an Fv or scFv. In embodiments, the Fv or scFv comprises an HCVR and a LCVR. In embodiments, the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3. In embodiments, the LCVRBT Ref.91016- 420148 DFCI3434.WO01WO comprises the CDRs LCDR1, LCDR2, and LCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0070] In embodiments, the antibody fragment comprises a VhH. In embodiments, the VhH comprises an HCVR. In embodiments, the HCVR comprises the CDRs HCDR1,BT Ref.91016- 420148 DFCI3434.WO01WO HCDR2, and HCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, and the amino acid sequence of HCDR3 is SEQ ID NO: 17. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, and the amino acid sequence of HCDR3 is SEQ ID NO: 23. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, and the amino acid sequence of HCDR3 is SEQ ID NO: 29. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, and the amino acid sequence of HCDR3 is SEQ ID NO: 35. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, and the amino acid sequence of HCDR3 is SEQ ID NO: 41. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, and the amino acid sequence of HCDR3 is SEQ ID NO: 47. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, and the amino acid sequence of HCDR3 is SEQ ID NO: 53.

[0071] Additional antibodies and fragments thereof that are useful in embodiments of the present disclosure can be found in US Patent Publication Nos. US10617773B2, US10059775B2, and US20230265208A1, each of which is incorporated by reference in its entirety on April 12, 2024.

[0072] Nucleic acids targeting MUC1-C may include DNA oligonucleotide or anti-sense RNA oligonucleotides (ASOs). By targeting MUC1-C DNA or RNA, ASO-mediated therapies may successfully target downstream pathways influencing the progression of premalignant lesions to SCC, or the progression of chronic inflammation inducing SCC transformation. Accordingly, any therapeutic agents targeting either MUC1-C at the DNA / RNA or protein level, and / or that inhibit expression and / or activity of MUC1-C, are contemplated as the therapeutic agents used in the methods described herein.

[0073] In embodiments, the MUC1-C inhibitor is DNA oligonucleotide or anti-sense RNA oligonucleotides (ASOs) or pharmaceutical composition thereof. In embodiments, the antibody or fragment thereof binds a MUC-1C nucleic acid sequence or MUC1-C target sequence. In embodiments, the MUC1-C inhibitor is a pharmaceutical composition,BT Ref.91016- 420148 DFCI3434.WO01WO comprising a (i) DNA oligonucleotide or anti-sense RNA oligonucleotides (ASOs), and (ii) one or more pharmaceutically acceptable carriers, diluents, or excipients. Target sequences of the MUC1-C are noted Table 2. Table 2. Target sequences of MUC1-C and antisense oligonucleotides against them

[0074] Another embodiment described herein is a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising administering a therapeutically effective amount of a composition comprising means for inhibiting MUC1-C expression or activity and a pharmaceutically acceptable excipient. In one aspect, the means may be a small molecule, a nucleic acid, a peptide or a biologic that targets MUC1-C at the DNA / RNA or protein level, and / or that inhibits expression and / or activity of MUC1-C, and their equivalents. Exemplary means include, but are not limited to, salinomycin, GO-203 peptide, antisense oligonucleotides, and anti-MUC1-C antibodies, such as 3D1 and 7B8, and other molecules known in the art to target MUC1-C, and equivalents thereof.

[0075] MUC1-C inhibitors may be formulated into any suitable formulation or composition for the intended application. Compositions for use in the methods described herein may be formulated into a dosage form that can be administered to a patient. In one aspect, the composition is in the form of an oral or parenteral dosage unit. In one aspect, it is in the form of an oral dosage unit. In one embodiment, the oral dosage unit is fractionated into several, smaller doses, which are administered to aBT Ref.91016- 420148 DFCI3434.WO01WO subject over a predetermined period of time to reduce toxicity of the therapeutic agent being administered. In another aspect, an oral dosage unit is administered as a tablet or capsule comprising a controlled release formulation that can include a plurality of particles, granules, pellets, mini-tablets or tablets. In another aspect, an oral dosage form is selected from a tablet, caplet, capsule, lozenge, syrup, liquid, suspension and elixir. In one aspect, an oral dosage form is selected from tablets, hard shell capsules, soft gelatin capsules, beads, granules, aggregates, powders, gels, solids and semi- solids.

[0076] In some cases, the composition is in the form of a parenteral dosage unit. For example, a parenteral dosage unit is selected from intravenous (IV), subcutaneous (SC), or transdermal dosage units. In another aspect, a dosage form is selected from sterile solutions, suspensions, suppositories, tablets and capsules. Since squamous cell carcinomas are localized, local injection of the MUC1-C inhibitor composition to an area suspected of or diagnosed as a premalignant lesion, may be a suitable mode of administration.

[0077] Many instances of squamous cell carcinomas involve the skin, thus compositions for use in the methods described herein also include dermatological compositions adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion. For example, dermatological compositions include a pharmaceutically or cosmetically acceptable medium. Dermatological compositions for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. In some cases, conventional pharmaceutical carriers, aqueous, powder or oily bases, skin enhancers, thickeners can be needed or desirable and therefore used. Suitable enhancers include ethers, e.g., diethylene glycol monoethyl ether (available as TRANSCUTOL®) and diethylene glycol monomethyl ether; surfactants, e.g., sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, Poloxamer (231, 182, 184), Tween (20, 40, 60, 80) and lecithin; alcohols, e.g., ethanol, propanol, octanol, benzyl alcohol; polyethylene glycol and esters thereof, e.g., polyethylene glycol monolaurate; amides and other nitrogenous compounds, e.g., urea, 2-pyrrolidone, l-methyl-2-pyrrolidone, dimethylacetamide (DMA), dimethylformamide (DMF), ethanolamine, diethanolamineBT Ref.91016- 420148 DFCI3434.WO01WO and triethanolamine; terpenes; alkanones; and organic acids, particularly citric acid and succinic acid. AZONE® and sulfoxides, e.g., DMSO and QOMSO may also be used, but are less preferred.

[0078]

[0164] In another aspect, the dosage form is selected from sustained release, controlled release, delayed release and response release forms.

[0079] The compositions and methods described herein have utility in treating many premalignant lesions and squamous cell carcinomas. In one aspect squamous cell carcinoma includes, but is not limited to, head and neck squamous cell carcinoma (HNSCC, including human papilloma virus (HPV)-negative HNSCC and HPV-positive HNSCC), skin or cutaneous squamous cell carcinoma (CSCC), adenoid squamous cell carcinoma, intra-epidermal squamous cell carcinoma, large cell keratinizing squamous cell carcinoma, lympho-epithelial carcinoma, papillary squamous cell carcinoma, or spindle cell carcinoma. In some aspects, squamous cell carcinoma may also include non-melanoma skin cancers. In another aspect, the squamous cell carcinoma is localized within the tissue, and may begin as a premalignant lesion in the tissue that may eventually progress to a squamous cell carcinoma. Thus, one embodiment described herein is a method of suppressing the progression of a premalignancy to a squamous cell carcinoma (SCC) in a patient in need thereof. Another embodiment is a method of suppressing chronic inflammation in a patient in need thereof. In one aspect, the squamous cell carcinoma is head and neck carcinoma (HNSCC). In another aspect, the squamous cell carcinoma is skin or cutaneous squamous cell carcinoma (CSCC).

[0080] In one aspect, the compositions and methods described herein are used as a first-line therapy (sometimes called primary therapy). In another aspect, the compositions and methods described herein are used as a second- line therapy. In some aspects, the compositions and methods described herein are used as third-line therapy. In some aspects, the compositions and methods described herein are used as a salvage therapy. The term “salvage therapy” means a therapeutic agent that can be taken with any regimen after a subject’s initial treatment regimen has failed or after the subject’s condition has not responded to an initial treatment. In another aspect, the compositions and methods described herein are used as a rescue therapy. In oneBT Ref.91016- 420148 DFCI3434.WO01WO embodiment, the compositions are used as a rescue agent to counteract the action of an initial treatment. In one embodiment, the compositions are used as rescue agent that is administered to a subject who has developed resistance to a standard or an initial treatment. In one aspect, the compositions and methods described herein are used as a neoadjuvant therapy. In one embodiment, a neoadjuvant therapy comprises administration of one or more of the therapeutic agents described herein to a subject before a main or first line treatment. In one embodiment, a neoadjuvant therapy reduces the size or extent of the cancer being treated before a main or first line treatment, primarily a surgery, is administered to the subject undergoing treatment. In another aspect, the compositions and methods described herein are used as an adjuvant therapy. In one embodiment, an adjuvant therapy comprises administration of one or more therapeutic agents described herein to a subject, wherein the one or more therapeutic agent that modify the effect of other therapeutic agents that are already administered to the subject or are concurrently administered to the subject or subsequently administered to the subject.

[0081] In one aspect, the compositions and methods of described herein exhibit toxicity levels that facilitate combinations with other pharmaceutical agents.

[0082] The methods and compositions described herein are not limited to a particular age of the subject. In one aspect, a subject or patient treated according to methods and using compositions described herein is less than 1 year old or older, and may include neonatal patients, pediatric patients, adult patients, or geriatric patients.

[0083] In one aspect, the patient has received at least one prior therapeutic agent. In one aspect the patient has received at least two, at least three, or at least four prior therapeutic agents. In another aspect the prior therapeutic agent is cisplatin, 5 - fluorouracil, pembrolizumab or nivolumab. In another aspect, the patient may have been previously treated with radiation. In one aspect, the cancer no longer responds to treatment with cisplatin, 5 -fluorouracil, pembrolizumab or nivolumab, radiation, surgery, or a combination thereof.

[0084] In one aspect, the methods described herein include administration of a second therapeutic agent includes an anti -cancer agent. In aspect, the second therapeutic agent is selected from cisplatin, 5 -fluorouracil, pembrolizumab, nivolumab orBT Ref.91016- 420148 DFCI3434.WO01WO combinations thereof.

[0085] In another aspect, the methods described herein include combining administration of the MUC1-C inhibitor composition with a surgical treatment. Exemplary surgical treatments include electrodessication and curettage, cryosurgery, Mohs micrographic surgery, excision surgery, laser surgery, photodynamic surgery, Mohs surgery, lymph node excision and biopsy, skin grafting and flap surgery. In some aspects the MUC1-C inhibiting compositions are administered as a neoadjuvant, prior to surgery. In other aspects, the MUC1-C inhibiting compositions are administered during and after surgery.

[0086] When used prior to or after surgery, the compositions described herein may be administered to a subject according to an infrequent dosing regimen (e.g., administered once per week or less frequently). In one aspect, the composition is administered to a subject according to a frequent dosing regimen (e.g., administered more than once per day or more than once per week). In another aspect, the composition is administered to a subject once weekly. In one aspect, the composition is administered to a subject once every four weeks. In one aspect, the composition is administered to a subject twice a week. In another aspect, the composition is administered to a subject once every two weeks. In one aspect, the composition is administered to a subject once every three weeks. In one aspect, the composition is administered to a subject in a repeated cycle of once daily, twice daily, once weekly, once every two weeks, once every three weeks, once every four weeks or combinations thereof. Any appropriate dosing regimen is contemplated herein.

[0087] Thus, another embodiment described herein is a method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising: (a) administering a first line therapeutic composition comprising a MUC1-C inhibitor; (b) performing a second line therapy comprising a surgery; wherein the first line therapy is administered local to the premalignant lesion; and wherein the second line therapy comprises an excision surgery to remove the premalignant lesion.

[0088] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readilyBT Ref.91016- 420148 DFCI3434.WO01WO apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. EXAMPLES Materials and Methods Cell culture

[0089] HPV-negative CAL27 cells (ATCC, RRID:CVCL_1107) were cultured in DMEM medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; GEMINI Bio-Products™, West Sacramento, CA, USA). HPV- negative HSC3 and FaDu cells ATCC, RRID:CVCL_1288 and 1218) were cultured in MEM medium (Corning®, Corning, NY, USA) supplemented with 10% FBS. Authentication of the cells was performed every 3-4 months by short tandem repeat (STR) analysis. Cells were monitored for mycoplasma contamination every 3-4 months using the MycoAlert®Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells were maintained for three months when performing experiments. Gene silencing

[0090] MUC1shRNA#1 (MISSION shRNA TRCN0000122938; Sigma, St. Louis, MO, USA) and a control scrambled shRNA (CshRNA; Sigma) were inserted into the pLKO- tet-puro vector (Plasmid #21915; Addgene, Cambridge, MA, USA) as described (See, Morimoto et al., 2023). The MUC1shRNA#2 (MISSION shRNATRCN0000430218) was produced in HEK293T cells (RRID:CVCL_0063) as described (See, Morimoto et al., 2023). Flag-tagged MUC1-C / CD was inserted into the empty control pLenti CMV Blast DEST(706-1) vector (Plasmid #17451; Addgene) as described (See, Yamashita et al., 2023). Vector-transduced cells were selected for growth in 1–2 μg / ml puromycin. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng / ml doxycycline (DOX; Millipore Sigma).BT Ref.91016- 420148 DFCI3434.WO01WO Immunoblot analysis

[0091] Total lysates prepared from non-confluent cells were subjected to immunoblot analysis using anti-MUC1-C (HM-1630-P1ABX, 1:200 dilution; ThermoFisher Scientific), anti-RIG-I (3743S, 1:1000 dilution; Cell Signaling Technology®(CST), Danvers, MA, USA), anti-MDA5 (5321S, 1:1000 dilution; CST), anti-STAT1 (9172S, 1:1000 dilution; CST), anti-STAT2 (72604S, 1:1000 dilution; CST), anti-IRF9 (76684S, 1:1000 dilution; CST), anti-IRF1 (8478S, 1:1000 dilution; CST), anti-OAS1 (14955-1-AP, 1:1000 dilution; Proteintech®, Rosemont, IL, USA), anti-MX1 (13750-1-AP, 1:1000 dilution; Proteintech®), anti-ISG15 (sc-166755, 1:1000 dilution; Santa Cruz Biotechnology®, Dallas, TX, USA), anti-GBP1 (15303-1-AP, 1:1000 dilution; Proteintech®), anti-IDO-1 (86630S, 1:1000 dilution, CST), anti-WARS (GTX110223, 1:1000 GeneTex, Irvine, CA), anti- ΔNp63 (619002, 1:1000 dilution; BioLegend®, San Diego, CA, USA), anti-SOX2 (3579, 1:1000 dilution; CST), anti-NOTCH3 (5276T, 1:1000 dilution, CST), anti-HEY1 (19929-1-AP, 1:2000 dilution; Proteintech®), anti-JAG1 (2620T, 1:1000 dilution, CST), anti-DLL3 (2483S, 1:1000 dilution, CST) and anti-b-actin (A5441, 1:10000 dilution; Sigma-Aldrich®, Burlington, MA, USA). Chromatin purified using the Chromatin Extraction Kit (ab117152, abcam, Cambridge, UK) was immunoblotted with anti-MUC1-C (HM-1630-P1ABX, 1:200 dilution; ThermoFisher Scientific), anti-STAT1 (9172S, 1:1000 dilution; CST) and anti- histone H3 (ab1791,1:5000 dilution, abcam). Colony formation assays

[0092] Cells were seeded in 24-well plates for 24 hours and then treated with (i) 0.1% DMSO or 500 ng / ml DOX, and (ii) PBS or GO-203. After 7-14 days, cells were stained with 0.5% crystal violet (LabChem, Zelienople, PA, USA) in 25% methanol. Growth was quantified at 590 nm using a spectrophotometer and normalized to DMSO treatment. Quantitative reverse-transcription PCR (qRT-PCR)

[0093] Total cellular RNA was isolated using Trizol reagent (ThermoFisher Scientific). cDNAs were synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Grand Island, NY, USA) as described (See, Morimoto et al., 2023).BT Ref.91016- 420148 DFCI3434.WO01WO The cDNA samples were amplified using the Power SYBR™Green PCR Master Mix (Applied Biosystems™) and the CFX96 Real-Time PCR System (BIO-RAD, Hercules, CA, USA) as described (See, Morimoto et al., 2023). Primers used for qRT-PCR are listed Table 3. Table 3. Primers used for qRT-PCR analysis FWD AGACGTCAGCGTGAGTGATG SEQ ID NO: 67BT Ref.91016- 420148 DFCI3434.WO01WO FWD GAAAACAATGCCCAGACTCAA SEQ ID NO: 91 ΔNp63 REVTGCGCGTGGTCTGTGTTA SEQ ID NO: 92Click-iT™Nascent RNA Assay

[0094] Nascent RNA labeling with ethylene uridine (EU) was performed using the Click- iT™Nascent RNA Capture kit(Invitrogen™, Waltham, MA, USA) as previously described (See, Bhattacharya et al., 2023). Cells were pulsed with 0.5 mM EU for 24 hours and nascent transcripts were captured from isolated total RNA on streptavidin magnetic beads. cDNA synthesis was performed on the beads using the High-Capacity cDNA Reverse Transcription Kit followed by qRT-PCR analysis. Chromatin immunoprecipitation (ChIP).

[0095] ChIP was performed on cells crosslinked with 1% formaldehyde for 10 min at 37°C, quenched with 2M glycine, washed with PBS, and sonicated in a Covaris®220 sonicator to generate 300-600 bp DNA fragments. Immunoprecipitation was performed using a control IgG (3900S, CST, RRID:AB_1550038) and antibodies against MUC1-C (16564S, CST, RRID:AB_2798765) and STAT1 (9172S, CST). Quantitation was performed on immunoprecipitated DNA using SYBR-green and the CFX384 real-time PCR machine (Bio-Rad, USA). Precipitated DNAs were detected by PCR using primers listed in Table 4. Data are reported as percentage of input DNA for each sample. Table 4. Primers used for ChIP-PCRBT Ref.91016- 420148 DFCI3434.WO01WO REV AGGTGGAAGTTGATGGATTGG SEQ ID NO: 100 FWDGTCCCATCCTCATTTAAGTACCC SEQ ID NO: 101Chromatin-bound protein extraction

[0096] Chromatin Extraction Kit (abcam) was used to isolate chromatin-bound proteins according to the manufacturer’s instructions. RNA-seq analysis

[0097] Total RNA from cells cultured in triplicates was isolated using Trizol reagent (Invitrogen) as described (See, Morimoto et al., 2023). TruSeq®Stranded mRNA (Illumina®, San Diego, CA, USA) was used for library preparation. Raw sequencing reads were aligned to the human genome (GRCh38.74) using STAR. Raw feature counts were normalized and differential expression analysis using DESeq2 as described (See, Morimoto et al., 2023). Differential expression rank order for subsequent Gene Set Enrichment Analysis (GSEA) was performed using the fgsea (v1.8.0) package in R. Hallmark Gene Sets were queried through the Molecular Signatures Database (MSigDB). Tumorsphere formation assays

[0098] Cells (5 x 103) were seeded per well in 6-well ultra-low attachment culture plates (Corning®Life Sciences, Tewksbury, MA, USA) in DMEM / F1250 / 50 medium (Corning Life Sciences) with 20 ng / ml EGF (Millipore Sigma, Boston, MA, USA), 20 ng / ml bFGF (Millipore Sigma) and 1% B27 supplement (Gibco™, Grand Island, NY, USA). Cells were treated with (i) 0.1% DMSO or 500 ng / ml DOX, and (ii) PBS or GO-203 as described (See, Morimoto et al., 2023). Tumorspheres were counted under an inverted microscope in triplicate wells. Example 1 Mouse tumor model studiesBT Ref.91016- 420148 DFCI3434.WO01WO Six-week-old nude mice (Jackson Laboratory, Bar Harbor, ME, USA) were injected subcutaneously into the flank with 1x107CAL27 cells in 100 ml of a 1:1 solution of medium and Matrigel®(BD Biosciences, Woburn, MA, USA). When the mean tumor volume reached 100–150 mm3, the mice were pair-matched into groups of five mice each. Mice were treated intraperitoneally each day with PBS or GO-203 at a dose of 12 μg / gm body weight. Tumor measurements and body weights were recorded twice per week. Tumor lysates were prepared using the T-PER tissue protein extraction reagent (#78510, ThermoFisher Scientific). The resource equation method was used for determining the minimum number of mice to achieve significance (See, Charan et al., 2013). Analysis of publicly available bulk RNA-seq HNSCC data

[0099] HNSCC RNAseq and clinical annotation dataset file of GSE136037 was downloaded from the Gene Expression Omnibus (GEO) database. GSE136037 included 49 primary lesions and 23 metastatic lesions. The downloaded data GSE13637 was used in read count format. Analysis of publicly available scRNA-seq HNSCC data

[0100] Data for publicly available scRNA-seq dataset of HNSCC samples (GSE181919) were obtained from GEO. GSE181919 included 20 primary tumor samples (13 HPV- negative and 7 HPV-positive). Expression (TPM) was obtained, with tumor cells identified using previously determined criteria (See, Choi et al., 2023). Uniform manifold approximation and projection (UMAP) representations of remaining tumor cells were produced from total expression profiles, with expression (TPM) compared between given factors. Generated census counts were obtained from GEO. Low quality cells previously determined were eliminated prior to downstream analysis. Remaining cells were analyzed by Seurat. Normalization and variance stabilization were conducted using regularized negative binomial regression (sctransform).BT Ref.91016- 420148 DFCI3434.WO01WO Statistical analysis

[0101] Each experiment was performed at least three times. Unpaired two-tailed Student’s t-tests were used to assess differences between the mean±SD of two groups. GraphPad Prism9 was used for all statistical analyses. P-values were considered significant at *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 with CI = 95%. Data Availability

[0102] Raw and processed RNA-seq data generated in this study are available from the NCBI Gene Expression Omnibus (GEO) under accession GSE252287. Results

[0103] Dependence of HNSCC cells on MUC1-C for clonogenicity. Analysis of the GSE136037 dataset demonstrated that MUC1 gene expression is significantly increased in metastatic vs primary HNSCC tumors (Fig.1A; Fig.2A). To investigate potential involvement of MUC1-C in HNSCC, HPV-negative CAL27 HNSCC cells expressing a CshRNA or MUC1shRNA vector were established. MUC1-C mRNA levels were decreased in CAL27 / MUC1shRNA, and not CAL27 / CshRNA, cells (Fig.1B, left). Moreover, the MUC1-C protein, which is expressed as glycosylated 25 kDa and unglycosylated 17 kDa forms, was decreased in CAL27 / MUC1shRNA cells (Fig.1B, right). It was found that silencing MUC1-C in CAL27 / MUC1shRNA cells suppresses their capacity for clonogenic survival (Fig.1C), demonstrating this oncoprotein is involved in HNSCC progression. MUC1-C consists of a 58 aa extracellular, 28 aa transmembrane and 72 aa cytoplasmic domain (See, Kufe et al., Cancer (Basel) 2022). The MUC1-C cytoplasmic domain (MUC1-CD) is an intrinsically disordered scaffold with nodes for integrating diverse signaling pathways (Fig.1D) (See, Kufe et al., Cancer (Basel) 2022). MUC1-CD is phosphorylated by EGFR, FGFR3 and MET (See, Kufe et al., Cancer (Basel) 2022). In addition, the MUC1-CD CQC motif, which is targeted by the GO-203 inhibitor, binds directly to JAK1 and the SAGNGGSSLS region associates with STAT1 in facilitating their interactions (Fig.1D) (See, Kufe et al., Cancer (Basel) 2022, and Khodarev et al., 2019). To further assess the effects of MUC1-C and MUC1- CD, an inducible tet-MUC1shRNA targeting a different region was used, which inBT Ref.91016- 420148 DFCI3434.WO01WO response to DOX treatment downregulated MUC1-C expression (Fig.1E). MUC1-C silencing was rescued by expressing a DOX-inducible Flag-MUC1-CD (tet-Flag-MUC1- CD) (Fig.1E). MUC1-CD reversed the suppressive effects of silencing MUC1-C on colony formation (Fig.1F). As an additional control, treatment of CAL27 / tet-CshRNA cells with DOX had little if any effect on MUC1-C expression and colony formation (Figs. 2B and 2C), showing that CAL27 cells are dependent on MUC1-CD for clonogenic survival. MUC1-C activates gene signatures associated with chronic inflammation

[0104] For comparison with CAL27 cells, human HPV-negative HSC3 (Figs.4A and 4B) and FaDu (Figs.4C and 4D) HNSCC cells expressing a tet-CshRNA or tet- MUC1shRNA were established and confirmed that MUC1-C expression is selectively downregulated in DOX-treated tet-MUC1shRNA cells. It was also found that, like CAL27 cells, silencing MUC1-C similarly suppresses clonogenicity of HSC3 and FaDu cells (Figs.4E and 4F). Volcano plots demonstrated that inducible MUC1-C silencing in CAL27 and HSC3 cells results in broad changes in down- and up-regulated gene expression (false discovery rate (FDR)<0.05, fold change (FC)>2)(Fig.3A). By comparing DEGs from CAL27 and HSC3 cells with MUC1-C silencing, (i) 441 common downregulated genes, which included STAT1 / 2, IRF9 and ISGs, and (ii) 138 upregulated genes were identified (Fig.3B). MUC1-C localizes to chromatin of cancer cells, where it interacts with TFs and effectors of epigenetic regulation (See, Kufe et al., Cancer (Basel) 2022, and Haratake et al., 2023). In concert with the findings that MUC1-C binds directly to STAT1 and regulates STAT1 target genes (See, Khodarev et al., 2010), analysis of purified chromatin in CAL27 cells demonstrated that silencing MUC1-C decreases expression of the MUC1-C 17 kDa monomer and 34 kDa homodimer in association with downregulation of STAT1 levels (Fig.3C). Assessment of the top MUC1-C-downregulated HALLMARK gene sets (Fig.4G) further identified the HALLMARK INTERFERON ALPHA (Fig.3D) and HALLMARK INTERFERON GAMMA RESPONSE (Fig.3E) signatures. Consistently, silencing MUC1-C in CAL27 and HSC3 cells was associated with suppression of downstream IFN type I / II pathway ISGs, whichBT Ref.91016- 420148 DFCI3434.WO01WO included (i) IRF7 and IRF9, (ii) OAS1-3 and OASL, (iii) MX1 / 2 and (iv) IFIT1 and IFITM1 (Figs.3F and 3G). MUC1-C drives intrinsic chronic inflammation by regulating the type I / II IFN pathways

[0105] The type I IFN pathway is chronically activated in cancer cells by the RIG-I and MDA5 PRRs that recognize cytosolic RNA (See, Khodarev et al., 2019; Li et al., 2018; and Mazewski et al., 2020). Silencing MUC1-C in CAL27 cells decreased RIG-I and MDA5 transcripts (Fig.5A) and protein levels, which were rescued with MUC1-CD (Fig. 5B). Congruent with driving these PRRs, silencing MUC1-C in CAL27 cells suppressed STAT1 / 2 and IRF9 (Figs.5C and 5D). Similar effects on PRR, STAT1 / 2 and IRF9 expression were observed in HSC3 cells with MUC1-C silencing (Supplemental Figs. 6A and 6B). STAT1 / 2 regulate expression of downstream ISGs (See, Michalska et al., 2018). Targeting MUC1-C is necessary for expression of OAS1, MX1 and ISG15 proteins that contribute to the IFN-related DNA damage signature (IRDS) (Figs.5E and 5F; Fig.6C) (See, Weichselbaum et al., 2008; Khodarev et al., 2012; Benci et al., 2016; and Sandy et al., 2020). Among these, OAS1 also amplifies the type I IFN pathway and expression of genes that promote immune evasion (See, Qiu et al., 2023). In addition, ISG15 links the DNA damage response to regulation of innate immunity (See, Wardlaw et al., 2023). Along these lines, we found that MUC1-C regulates expression of guanylate-binding protein 1 (GBP1), which is activated by chronic inflammation and confers treatment resistance (See, Honkala et al., 2020; Wu et al., 2020) (Figs.5G and 5H; Fig.6D). Silencing MUC1-C also decreased expression of (i) IDO1, which suppresses tryptophan (Trp) levels in the tumor microenvironment (TME) for T cell function (See, Prendergast et al., 2017), and (ii) tryptophanyl-tRNA synthetase (WARS, WRS) that protects cancer cells from Trp depletion (See, Adam et al., 2018; Ahn et al., 2021)(Figs.5G and 5H). Similar results were obtained when targeting MUC1-C in HSC3 cells which, unlike CAL27 cells, have undetectable levels of IDO1 expression (Fig.6D). These findings show a role for MUC1-C in regulating the type I and II IFN pathways in HNSCC cells and integrating expression of ISGs that promote DNA damage resistance and immune evasion associated with the CSC state (See, Kufe et al., Cancer (Basel)BT Ref.91016- 420148 DFCI3434.WO01WO 2022, Yamashita et al., 2023; Miranda et al., 2019; De Angelis et al., 2019, Quintanal- Villalonga et al., 2020; Crum et al., 2010). MUC1-C and STAT1 regulate ΔNp63 expression

[0106] The ΔNp63 TF plays a role in (i) homeostasis of the epidermis, (ii) SCC pathogenesis, and (iii) self-renewal of CSCs(See,Gatti et al., 2019, Moses et al., 2019; Pecorari et al., 2022; Fisher et al., 2023). There is no known association between MUC1-C and ΔNp63. Targeting MUC1-C decreased ΔNp63 gene transcription and mRNA levels in CAL27 and HSC3 cells (Fig.7A; Fig.8A). Silencing STAT1 suppressed ΔNp63 expression (Fig.7B; Fig.8B), showing the MUC1-C / STAT1 auto-inductive pathway was involved (See, Khodarev et al., 2010). ΔNp63 is transcribed from a promoter in the third intron of the TP63 gene (See, Pecorari et al., 2022); a promoter like signature (PLS) in this region contains a potential STAT binding motif (Fig.7C). In concert with MUC1-C binding to STAT1 in regulating STAT1 target genes (See, Khodarev et al., 2010), ChIP studies of the PLS demonstrated that MUC1-C and STAT1 occupy this region and that silencing MUC1-C decreases their occupancy (Fig.7D). Consistent with these results, targeting MUC1-C and STAT1 downregulated expression of the ΔNp63 protein (Figs.7E and 7F); Figs.8C and 8D), showing that MUC1- C / STAT1 signaling is involved in ΔNp63 expression. MUC1-C and STAT1 regulate SOX2 expression

[0107] SOX2 is involved in self-renewal of basal cells, which are the origin of SCCs(See, Moses et al., 2010; Crum et al., 2010). In addition, SOX2 contributes to progression of the SCC CSC state (See, Singh et al., 2021; Boumahdi et al., 2014). As found for ΔNp63, targeting MUC1-C in CAL27 and HSC3 cells downregulated SOX2 gene transcription and mRNA levels (Fig.9A; Fig.10A). Silencing STAT1 also decreased activation of the SOX2 gene (Fig.9B; Fig.10B), showing that MUC1- C / STAT1 complexes drive SOX2 expression. STAT1 binding motifs were identified in a SOX2 PLS (Fig.9C) that were occupied by STAT1 in a MUC1-C-dependent manner (Fig.9D). Targeting MUC1-C and STAT1 also decreased expression of the SOX2BT Ref.91016- 420148 DFCI3434.WO01WO protein (Figs.9E and 9F), showing that MUC1-C / STAT1 signaling integrates regulation of the ΔNp63 and SOX2 genes in HNSCC cells. Targeting MUC1-C downregulates NOTCH3 and HNSCC cell self-renewal Capacity

[0108] Functional and genomic analyses have identified an important role for the NOTCH3 stemness factor in HNSCC progression (See, Kondratyev et al., 2023). Silencing MUC1-C in CAL27 and HSC3 cells decreased expression of (i) NOTCH3, (ii) jagged canonical ligand 1 (JAG1), (iii) the delta-like ligand 3 (DLL3), and (iv) downstream NOTCH target HEY1 (See Kondratyev et al., 2023; You et al., 2023) (Fig. 11A; Fig.12A). Concordant with these results, silencing MUC1-C suppressed the GOBP NOTCH SIGNALING PATHWAY and REACTOME SIGNALING BY NOTCH gene signatures (Fig.11B; Fig.12B). Dysregulation of ΔNp63, SOX2 and NOTCH3 have each been linked to HNSCC cell stemness (See Dotto et al., 2016). Consistent with MUC1-C dependence for ΔNp63, SOX2 and NOTCH3 expression, silencing MUC1-C in CAL27 and HSC3 cells decreased self-renewal capacity as evidenced by suppression of tumorsphere formation (Fig.11C; Fig.12C). Moreover, it was confirmed that MUC1-CD recovers sphere forming efficiency (SFE) (Fig.11C; Fig.12C). The 72 aa MUC1-C cytoplasmic domain includes a CQC motif that facilitates MUC1-C dimerization and function (Fig.1C) (See, Kufe et al., Cancer(Basel) 2022). Targeting the MUC1-C CQC motif with the GO-203 inhibitor, which blocks MUC1-C nuclear localization and function (See, Kufe et al., Cancer(Basel) 2022) decreased ΔNp63, SOX2 and NOTCH3 expression (Fig.11D; Fig.12D). GO-203 treatment of CAL27 (Fig.13E) and HSC3 (Fig. 12E) cells also suppressed colony formation, confirming dependence of HNSCC cells on MUC1-C for clonogenic survival. In addition, targeting MUC1-C with GO-203 significantly inhibited SFE of CAL27 (Fig.11F) and HSC3 (Fig.12F) cells, demonstrating that MUC1-C regulates their self-renewal capacity. GO-203 treatment of mice with established CAL27 xenografts further demonstrated dependence on MUC1-C for tumorigenicity (Fig.11G). Immunoblot analysis of CAL27 tumors from control and GO-203-treated mice show that targeting MUC1-C downregulates ΔNp63 and SOX2 expression (Fig.11H).BT Ref.91016- 420148 DFCI3434.WO01WO MUC1 expression in HNSCC tissues

[0109] To extend findings to HNSCC tumors, GSE181919 scRNA-seq dataset derived from 20 primary HNSCCs and 4 HNSCCs metastatic to lymph nodes were analyzed (See, Choi et al., 2023). HNSCC cells were distinguished from non-malignant cell types by the presence of DNA copy number aberrations (CNAs) (See, Choi et al., 2023; Woo et al., 2023). Analysis of malignant HNSCC cells with recurrent CNAs demonstrated higher levels of MUC1 expression compared to that in other cell types (Fig.13A). The GSE181919 dataset includes HPV-negative and HPV-positive HNSCCs (See, Choi et al., 2023) which differ in DNA methylation and gene expression profiles (See, Burkitt et al., 2023). CAL27 and HSC3 cells studied here are HPV-negative; accordingly, HPV- negative HNSCCs in the GSE181919 dataset were analyzed and showed similar upregulation of MUC1 expression in the malignant cell population (Fig.13B). Analysis of the HPV-negative / HPV-positive (Fig.14A) and HPV-negative (Fig.13C) HNSCC data also showed expression of STAT1, ΔNp63, SOX2 and NOTCH3 in HNSCC cells and, to a variable extent, in other cell populations. Further analysis of malignant HNSCC cells in the HPV-negative / HPV-positive (Fig.14B) and HPV-negative (Fig.14C) datasets uncovered significant correlations of MUC1 expression with ΔNp63, SOX2 and NOTCH3. By contrast, correlations of MUC1 with STAT1 were not significant in terms of their transcripts. Of further interest, analysis of HPV-positive HNSCC tumors demonstrated MUC1 expression in the malignant cell population (Fig.14D), showing MUC1-C is also a target for HPV-positive HNSCCs.

[0110] MUC1-C contributes to HNSCC cell intrinsic pathways of chronic inflammation. In this way, MUC1-C regulates expression of the RIG-I and MDA5 PRRs that are activated by cytosolic RNA (See, Khodarev et al., 2019; Li et al., 2018; Mazewski et al., 2020). MUC1-C promotes DNA replicative stress; however, a role in increasing cytosolic RNA is not known (See, Kufe et al., Cancer (BaseI) 2023). Nonetheless, RIG-I and MDA5 contribute to intrinsic upregulation of IFNb production and the type I IFN pathway (See, Yamashita et al., 2022). MUC1-C binds to STAT1 and regulates transactivation of STAT1 target genes (See, Khodarev et al., 2010). In concert with this function and the role of STAT1 in activation of the type I / II IFN pathways, it was found that MUC1-CBT Ref.91016- 420148 DFCI3434.WO01WO regulates intrinsic activation of downstream ISGs, including OAS1, MX1 and ISG15 that are effectors of DNA damage resistance and immune evasion (Fig.13D) (See, Khodarev et al., 2019). Barrier tissues are dependent on resident stem cells (SCs) that, following injury, are activated to promote wound healing (See, Dekoninck et al., 2019). SC lineage infidelity occurs transiently in wound healing, but persists in settings of chronic inflammation that promote cancer progression (See, Watanabe et al., 2014). The results uncover the previously unrecognized findings that MUC1-C-induced chronic activation of the STAT1 inflammatory pathway in HNSCC cells regulates the lineage- dictating ΔNp63 and SOX2 genes, which are amplified in HNSCCs and contribute to HNSCC pathogenesis (Fig.13D) (See, Watanabe et al., 2014). Mechanistically, it was found that MUC1-C and STAT1 occupy the ΔNp63 gene and that silencing MUC1-C decreases STAT1 occupancy and ΔNp63 expression. ΔNp63 is coamplified with SOX2 in SCCs and the ΔNp63 and SOX2 proteins form a direct complex that regulates SCC gene expression (See, Watanabe et al., 2014). Like ΔNp63, it was found that the SOX2 gene is occupied by MUC1-C and STAT1. MUC1-C was also necessary for STAT1 occupancy of SOX2 and targeting MUC1-C and STAT1 downregulated SOX2 expression.

[0111] These surprising results show MUC1-C / STAT1 complexes regulate ΔNp63 and SOX2, which are necessary for driving the HNSCC CSC state (Fig.13D) (See, Watanabe et al., 2014). By extension and in addition to ΔNp63 and SOX2, it was found that MUC1-C regulates expression of NOTCH3, which regulates HNSCC cell self- renewal and progression (See, Kondratyev et al., 2023). ΔNp63, SOX2 and NOTCH3 have each been shown to play a role in conferring the HNSCC CSC state (See, Watanabe et al., 2014). Currently, there is no evidence for a common pathway that integrates these TFs in driving HNSCC pathogenesis. Our results support a model in which MUC1-C regulates intrinsic chronic inflammation in HNSCC cells through activation of STAT1 and the IFN type I / II pathways (Fig.13D). In this way, targeting MUC1-C genetically and pharmacologically with the GO-203 inhibitor regulates downstream ISGs that contribute to DNA damage resistance and immune evasion (Fig. 13D). The results also support a previously unreported role for MUC1-C / STAT1 signaling in integrating chronic inflammation with regulation of (i) lineage-dictatingBT Ref.91016- 420148 DFCI3434.WO01WO ΔNp63 and SOX2 TFs, and (ii) NOTCH3 and self-renewal capacity (Fig.13D). Collectively, these findings and the demonstration that MUC1 associates with expression of ΔNp63, SOX2 and NOTCH3 in individual HNSCC tumor cells indicate that MUC1-C is a common effector of the HNSCC CSC state (Fig.13D). Example 2

[0112] The experiments described above were similarly performed in cutaneous squamous cell carcinoma cells. The results obtained from human SCC13 cutaneous SCC cells as depicted in Figures 15 – 18 demonstrate that targeting MUC1-C suppresses (i) ΔNp63 and NOTCH expression, and (ii) self-renewal capacity as determined by tumorsphere formation.

[0113] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. References provided throughout are meant to be incorporated by reference only with regard to such background teaching that they refer to. EQUIVALENTS

[0114] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

BT Ref.91016- 420148 DFCI3434.WO01WO WHAT IS CLAIMED:

1. A method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising administering a therapeutically effective amount of a MUC1-C inhibitor.

2. The method of claim 1, wherein the squamous cell carcinoma is a head and neck SCC or a cutaneous SCC.

3. The method of claims 1 or 2, wherein the squamous cell carcinoma is localized to a tissue.

4. The method of claims 1 – 3, wherein the MUC1-C inhibitor is a peptide, a small molecule, a biologic, or a nucleic acid.

5. The method of claim 4, wherein the inhibitor is a GO-203 peptide.

6. The method of claim 4, wherein the inhibitor is salinomycin.

7. The method of claim 4, wherein the inhibitor is an anti-sense RNA oligonucleotide.

8. The method of claims 1 – 7, wherein the inhibitor is formulated as a topical or an injectable.

9. The method of claims 1 – 8, wherein administration is localized.

10. The method of claims 1 – 9, wherein administration is prior to a surgical excision of the SCC.

11. The method of claims 1 – 10, further comprising administration of one or more of a chemotherapy, immunotherapy, or a surgical excision of the SCC.BT Ref.91016- 420148 DFCI3434.WO01WO 12. The method of claim 11, wherein chemotherapy comprises one or more of a cisplatin or 5-fluorouracil.

13. The method of claim 11, wherein the immunotherapy comprises one or more of cetuximab, pembrolizumab or nivolumab.

14. A method of suppressing chronic inflammation in a patient in need thereof comprising administering a therapeutically effective amount of a MUC1-C inhibitor.

15. A method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising administering a therapeutically effective amount of a composition comprising means for inhibiting MUC1-C expression or activity and a pharmaceutically acceptable excipient.

16. A method of suppressing the progression of a premalignant lesion to a squamous cell carcinoma (SCC) in a patient in need thereof comprising: (a) administering a first line therapeutic composition comprising a MUC1-C inhibitor; (b) performing a second line therapy comprising a surgery; wherein the first line therapy is administered local to the premalignant lesion; and wherein the second line therapy comprises an excision surgery local to the premalignant lesion.

17. The method of claim 16, wherein the squamous cell carcinoma is a head and neck squamous cell carcinoma or a cutaneous squamous cell carcinoma.

18. The method of claims 16 -17, wherein the first line therapeutic composition is administered according to a dosing regimen.BT Ref.91016- 420148 DFCI3434.WO01WO 19. A method of treating recurrent or progressive squamous cell carcinoma, comprising administering to a patient in need thereof an effective amount of (a) a MUC1-C inhibitor disclosed herein, or (b) a pharmaceutical composition thereof.

20. The method of claim 19, wherein the pharmaceutical composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

21. Use of (a) a MUC1-C inhibitor disclosed herein, or (b) a pharmaceutical composition thereof for treating recurrent or progressive squamous cell carcinoma.

22. The use of claim 21, wherein the pharmaceutical composition comprises a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

23. A MUC1-C inhibitor disclosed herein or pharmaceutical composition thereof for use in therapy.

24. The pharmaceutical composition of claim 23, comprising a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

25. A MUC1-C inhibitor disclosed herein or pharmaceutical composition thereof for use in treating recurrent or progressive squamous cell carcinoma.

26. The pharmaceutical composition of claim 25, comprising a dermatological composition adapted for cutaneous topical administration local to an area suspected of or diagnosed as a premalignant lesion.

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