Figure S2. Hsc70 binds and colocalises with TBX3 in FLAG-TBX3 (1,2) overexpressing and parental MCF-7 cells in vivo.

Figure S1: Establishment of FLAG-TBX3 ER+ breast cancer cells for mass spectrometry.

Background: Melanoma and triple-negative breast cancer (TNBC) are the most aggressive skin and breast cancers, often diagnosed at late stages with limited treatment options. The melanoma-associated antigen melanotransferrin (MTf) is overexpressed in these solid tumors, where it drives tumorigenesis, progression, and chemoresistance. Its inhibition correlates with tumor regression, making MTf a promising therapeutic target. This study aimed to develop a novel, selectively targeted antibody–drug conjugate (ADC) against MTf-expressing melanoma and TNBC cancer cells using SNAP-tag fusion protein conjugation technology. Methods: We generated an L49(scFv)-SNAP-tag antibody fusion protein engineered through the genetic fusion of a humanized anti-MTf single-chain variable fragment (scFv) with a SNAP-tag fusion protein capable of site-specific self-labelling with O6-benzylguanine (BG) modified substrates in 1:1 stoichiometry. Binding and internalization of the conjugate labeled with BG-Alexa 488 (L49(scFv)-SNAP-Alexa488) were assessed by confocal microscopy and flow cytometry in MTf-overexpressing cell lines. Cytotoxicity was evaluated using the cell viability XTT assay after conjugating the SNAP-fusion protein to the potent monomethyl auristatin-F (BG-AURIF). Results: The L49(scFv)-SNAP-Alexa488 conjugate demonstrated specific binding and internalization into MTf-positive melanoma and TNBC cells. The corresponding ADC, L49(scFv)-SNAP-Linker-AURIF, exerted potent, antigen and dose-dependent cytotoxicity, with IC50 values in the nanomolar range (4.77–34.43 nM). Conclusions: We successfully generated a novel SNAP-tag-based ADC that selectively eliminates MTf-overexpressing tumor cells. This proof-of-concept highlights MTF’s value as a therapeutic target and demonstrates that a smaller-format, non-cleavable linker SNAP-tag-based ADC can achieve potent nanomolar cytotoxicity, supporting further development of MTF-targeted immunotherapies for melanoma and TNBC.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that has a tropism for endothelial cells and leads to the development of Kaposi’s sarcoma, especially in people living with HIV. The present study aimed to quantify morphological and mechanical changes in endothelial cells after infection with KSHV to assess their potential as diagnostic and therapeutic markers. Vascular (HuARLT2) and lymphatic endothelial cells (LEC) were infected with recombinant KSHV (rKSHV) by spinoculation, establishing stable infections (HuARLT2-rKSHV and LEC-rKSHV). Cellular changes were assessed using mitochondria-tracking microrheology and morphometric analysis. rKSHV infection increased cellular deformability, indicated by higher mitochondrial mean squared displacement (MSD) for short lag times. Specifically, MSD at τ = 0.19 s was 49.4

ABSTRACT Background Melanoma and triple negative breast cancer (TNBC) represent the most aggressive skin and breast cancer subtypes and are associated with poor diagnostic and limited therapeutic options leading to poor prognosis. Melanotransferrin/p97 (MTf), initially identified as a tumor‐associated antigen (TAA) in melanoma, is overexpressed in various solid tumors, including TNBC. Beyond its high differential expression and dreadful tumorigenic impact, MTf is also associated with chemoresistance development, and its inhibition significantly hampers tumor progression, making MTf a promising target for effective targeted therapies. Near‐infrared photoimmunotherapy (NIR‐PIT) is an approach that combines the precision of antibodies directed against specific TAA with the phototoxic effects of a light‐sensitive photosensitizer (IR700), activated by near‐infrared (NIR) light irradiation. This study aimed to generate a novel photoimmunoconjugate to specifically destroy MTf‐positive melanoma and TNBC cells in vitro following NIR light irradiation. Methods A single‐chain variable fragment (scFv) assembled from anti‐MTf antibody L49 was recombinantly fused with the SNAP‐tag protein (L49(scFv)‐SNAP), capable of irreversible and autocatalytic conjugation to any O(6)‐benzylguanine (BG) substrate in a 1:1 stoichiometry. Purified full‐length SNAP‐tag–based fusion protein (L49(scFv)‐SNAP‐tag) was either conjugated to a BG‐modified fluorescent imaging agent (Alexa 488) to specifically assess its selective binding to MTf‐expressing cell lines via confocal imaging and flow cytometry or to a BG‐modified light‐sensitive photosensitizer (IR700) to evaluate its phototoxic properties using an XTT cell viability assay. Results The selective binding and internalization of L49(scFv)‐SNAP‐Alexa 488 towards MTf‐positive melanoma and TNBC cell lines were successfully demonstrated with MTF expression percentages ranging from 52.8 to 83.1. Once confirmed, dose‐dependent phototoxicity of L49(scFv)‐SNAP‐IR700 was achieved on illuminated MTf‐positive cell lines showing IC50 values in the nanomolar range (2.20–5.24 nM). Conclusion This study highlights the therapeutic potential of MTf as a promising target for the diagnosis as well as selective and efficient elimination of NIR‐light‐accessible melanoma and TNBC by NIR‐PIT. Trial Registration NCT03769506

Breast cancer is the second leading cause of death in women globally, and it remains a health burden due to poor therapy response, cancer cell drug resistance, and the debilitating side effects associated with most therapies. One approach to addressing the need to improve breast cancer therapies has been to elucidate the mechanism(s) underpinning this disease to identify key drivers that can be targeted in molecular therapies. The T-box transcription factor, TBX3, is upregulated in breast cancer, in which it contributes to important oncogenic processes, and it has been validated as a potential therapeutic target. Here, we investigated the molecular mechanisms that upregulate TBX3 in breast cancer, and we show that it involves transcriptional activation by c-Myc, post-translational modification by AKT1 and AKT3, and interaction with the molecular chaperone Hsc70. Together, the results from this study provide evidence that c-Myc, AKT, Hsc70, and TBX3 form part of an important oncogenic pathway in breast cancer and thus reveal versatile ways of interfering with the oncogenic activity of TBX3 for the treatment of this neoplasm. Implications: Targeting the c-Myc/AKT/TBX3/Hsc70 signaling axis may be an effective treatment strategy for TBX3-driven breast cancer.

Many animals and plants show left-right (LR) asymmetry. In some animal systems, handedness has a simple genetic basis, which has allowed identifying how handedness is determined at the molecular level, even if its functional relevance remains unclear. Mirror-image flowers represent an example of LR asymmetry of clear functional significance, with the reciprocal placement of male and female organs in left-versus right-handed flowers promoting cross-pollination. Here, we use the South African geophyte Cyanella alba to study how handedness of its mirror-image flowers is determined and elaborated during development. Inflorescences of C. alba produce flowers with a largely consistent handedness. However, we find that this handedness has no simple genetic basis, and individual plants can switch handedness between years. Rather, it is the direction of the phyllotactic spiral that determines floral handedness. Cellular analysis combined with biophysical modelling demonstrates that style deflection is driven by increased cell expansion in the adaxial carpel facing the next oldest flower compared to the other adaxial carpel. The carpel with greater expansion shows transcriptional signatures of increased auxin signaling and cell-wall modifications compared to the less expanding one. We propose that a recently described inherent LR auxin asymmetry in the initiating organs of spiral phyllotaxis determines handedness in C. alba , representing a conserved non-genetic mechanism for creating a stable floral polymorphism. This mechanism links chirality across different levels of plant development and exploits a developmental constraint in a core patterning process to produce morphological variation of ecological relevance. SIGNIFICANCE STATEMENT Several plant species show left-right asymmetry in their flowers, with the female reproductive organ displaced to the left or to the right of the midline, and at least one of the male organs positioned reciprocally. This increases reproductive success by promoting cross-pollination between individuals, yet how the handedness of flowers is established is unknown. Here, we show that most Cyanella alba plants form flowers of only one handedness, but this is not genetically controlled. Rather, left-right determination in flowers builds on a patterning process at the shoot tip that forms leaf and flower primordia in a clockwise or counter-clockwise spiral. Thus, an inbuilt handedness at one level of development is used to establish left-right asymmetry of ecological relevance. ### Competing Interest Statement The authors have declared no competing interest.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.

Body mass graphs from in vivo studies showing no change in body weight over the treatment time-courses.

Supplemental Figure Legends

IC50 values obtained from INI-43 treatment of cancer, transformed and normal cell lines.

Dose-response curves showing cell line responses to INI-43 treatment.

Cell stiffness and T-box transcription factor 3 (TBX3) expression have been identified as biomarkers of melanoma metastasis in 2D environments. This study aimed to determine how mechanical and biochemical properties of melanoma cells change during cluster formation in 3D environments. Vertical growth phase (VGP) and metastatic (MET) melanoma cells were embedded in 3D collagen matrices of 2 and 4 mg/ml collagen concentrations, representing low and high matrix stiffness. Mitochondrial fluctuation, intracellular stiffness, and TBX3 expression were quantified before and during cluster formation. In isolated cells, mitochondrial fluctuation decreased and intracellular stiffness increased with increase in disease stage from VGP to MET and increased matrix stiffness. TBX3 was highly expressed in soft matrices but diminished in stiff matrices for VGP and MET cells. Cluster formation of VGP cells was excessive in soft matrices but limited in stiff matrices, whereas for MET cells it was limited in soft and stiff matrices. In soft matrices, VGP cells did not change the intracellular properties, whereas MET cells exhibited increased mitochondrial fluctuation and decreased TBX3 expression. In stiff matrices, mitochondrial fluctuation and TBX3 expression increased in VGP and MET, and intracellular stiffness increased in VGP but decreased in MET cells. The findings suggest that soft extracellular environments are more favourable for tumour growth, and high TBX3 levels mediate collective cell migration and tumour growth in the earlier VGP disease stage but play a lesser role in the later metastatic stage of melanoma. Symbols

SubG1 cell populations after INI-43 treatment of CaSki cells for 6 and 24 hr.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III β subunit (β clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted β clamp-binding motif abolishes ImuB-β clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this β clamp-binding antibiotic collapses pre-formed ImuB-β clamp complexes. These observations establish the essentiality of the ImuB-β clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

Chemical compounds identified in the in silico screen that display IC50 values of less than 50uM.

Purpose Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody–drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. Methods Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. Results After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. Conclusion This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

During chemotherapy, structural and mechanical changes in malignant cells have been observed in several cancers, including leukaemia and pancreatic and prostate cancer. Such cellular changes may act as physical biomarkers for chemoresistance and cancer recurrence. This study aimed to determine how exposure to paclitaxel affects the intracellular stiffness of human oesophageal cancer of South African origin in vitro. A human oesophageal squamous cell carcinoma cell line WHCO1 was cultured on glass substrates (2D) and in collagen gels (3D) and exposed to paclitaxel for up to 48 h. Cellular morphology and stiffness were assessed with confocal microscopy, visually aided morpho-phenotyping image recognition and mitochondrial particle tracking microrheology at 24 and 48 h. In the 2D environment, the intracellular stiffness was higher for the paclitaxel-treated than for untreated cells at 24 and 48 h. In the 3D environment, the paclitaxel-treated cells were stiffer than the untreated cells at 24 h, but no statistically significant differences in stiffness were observed at 48 h. In 2D, paclitaxel-treated cells were significantly larger at 24 and 48 h and more circular at 24 but not at 48 h than the untreated controls. In 3D, there were no significant morphological differences between treated and untreated cells. The distribution of cell shapes was not significantly different across the different treatment conditions in 2D and 3D environments. Future studies with patient-derived primary cancer cells and prolonged drug exposure will help identify physical cellular biomarkers to detect chemoresistance onset and assess therapy effectiveness in oesophageal cancer patients.