Background/Rationale: PTK6/Brk, a non-receptor tyrosine kinase, is highly expressed in approximately 40% of triple negative breast cancers (TNBC). Higher levels of PTK6 expression are associated with worse patient outcomes. PTK6 is a driver of oncogenic growth, survival, invasion, epithelial-mesenchymal transition (EMT), chemotherapy resistance and metastasis. Recent studies have reported an association between PTK6 expression and an immunosuppressive microenvironment in patient breast cancers. Therefore, in addition to tumor cell-intrinsic oncogenic functions, PTK6 may play a critical role in shaping the tumor immune microenvironment which could modulate sensitivity to chemo/immunotherapy of TNBC. Inhibition of PTK6 could therefore suppress TNBC tumor growth and metastasis via dual (cell autonomous and microenvironmental) mechanisms. Methods. Genetic and pharmacological approaches were used to downregulate/inhibit PTK6 in immunocompetent and immunodeficient mouse models of TNBC (MMTV-myc, 4T-1). The effects of PTK6 shRNA expression, as well as treatment with a validated PTK6 kinase inhibitor (P21d), on TNBC tumor growth were monitored. For studies using immunocompetent models, immune populations (tumor-infiltrating and systemic) were analyzed by flow cytometry and immunofluorescence. Results: MMTV-myc or 4T-1 tumor-bearing, immunocompetent mice were treated with P21d, a validated small molecule inhibitor of PTK6 kinase activity. P21d treatment significantly inhibited tumor growth and increased tumoral infiltration by activated, cytotoxic immune cells (CD8+ T cells, NK cells). These effects were phenocopied by PTK6 shRNA or SNAIL shRNA expression, both of which also promote EMT reversal. Interestingly, the effects of P21d and PTK6 shRNA treatment on tumor growth were discordant when these same tumors were grown in immunodeficient mice; PTK6 shRNA, but not P21d treatment, inhibited TNBC growth. These results indicate that P21d’s inhibitory effect on tumor growth is reliant on immune modulation and that this immune regulation is PTK6 kinase-activity dependent. These results also suggest that there are kinase activity-independent mechanisms that are important for tumor growth control by PTK6. The importance of immune modulation in P21d-dependent triple negative tumor inhibition was reinforced by the fact that co-treatment of tumors with CD8 T cell or NK cell-depleting antibodies nearly completely abrogated P21d's effects on tumor growth. We identified CXCL10 as a mediator of P21d treatment-induced tumoral immune cell infiltration and tumor growth inhibition. Summary/Conclusion: Our studies highlight novel tumor immune microenvironmental functions of PTK6 oncogene in TNBC. PTK6 inhibition leads to recruitment and activation of cytotoxic T and NK cells that are critical for TNBC growth inhbition. Ongoing studies will determine whether these functions can be leveraged to enhance efficacy of immunotherapies currently used in the care of patients with TNBC. Citation Format: Hanna Irie, Ibuki Harada, Criseyda Martinez, Koichi Ito, Eunjee Lee, Jun Zhu. Inhibition of oncogenic PTK6 kinase enhances immune response against triple negative breast cancer via EMT reversal [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-06-17.

We introduce a new hydrological index that enables assessment of extreme events every few days from the GRACE Follow-On (GRACE-FO) satellite mission. The Mass Change Index (MCI) was developed by standardizing instantaneous satellite gravity anomalies computed directly from orbit perturbations. It is based on hydrology-related gravity change, namely, total water storage change, and thus equally sensitive to wet and dry anomalies. The key innovation of MCI is its sensitivity to instantaneous mass changes as opposed to monthly mean changes. GRACE-FO's ground track permits MCI retrievals every 5-6 days in most low and mid latitude regions. We demonstrate the application of MCI to investigate hydrological extremes in the middle-lower Yangtze River Basin (MLYRB). MCI detects extreme wet conditions (standardized index of 2.0-3.0) along the Yangtze River mainstream related to the catastrophic flood in 2020, consistent with daily streamflow observations. In contrast, a typical GRACE-FO based monthly drought index significantly underestimates the severity of the event and misidentifies timing of the onset. MCI also detects extreme dry conditions (-2.0 to -2.5) prevailing within MLYRB, related to the unprecedented heatwave and drought event during the summer of 2022. A streamflow index and the monthly drought index both underestimate the severity of the event. MCI retains information in intersatellite range measurements that may be lost when processing monthly gravity solutions. It can also be processed more rapidly, increasing its potential value for hydrological monitoring systems and other operational applications.

The non-receptor tyrosine kinase PTK6 is expressed in 70% of triple negative breast cancers (TNBC) and is an oncogenic driver of epithelial-mesenchymal transition (EMT). EMT promotes metastasis and immune evasion of TNBC. Therefore, targeting EMT drivers could reverse these properties and lead to more favorable outcomes. Treatment of TNBC tumors with a small molecule inhibitor of PTK6 kinase (P21d) suppressed their growth in vivo. Tumor inhibition by P21d is dependent on an induced immune response because: 1) inhibition is observed in immunocompetent, but not immunodeficient, mice; 2) P21d increases tumor-infiltrating CD8+ T and NK cells and decreases immunosuppressive myeloid-derived suppressor cells; and 3) tumor inhibition by P21d is abrogated by co-treatment with NK or CD8+ T cell-depleting antibodies. These effects on tumor growth and cytotoxic TILs are phenocopied by the knockdown of tumoral PTK6 or SNAIL, which supports EMT inhibition as a mechanism for enhanced anti-tumor immune response. RNA sequencing (RNA-seq) profiling of P21d-treated tumors also revealed changes consistent with activation of the immune response and identified CXCL10 as a critical chemokine induced intratumorally by P21d that promotes recruitment of NK/CD8+ T cells to the tumor site, leading to tumor growth inhibition. Our study highlights the novel tumor immune microenvironmental functions of PTK6 with important consequences for tumor growth that could lead to new immunotherapeutic approaches for TNBC. ### Competing Interest Statement The authors have declared no competing interest.

In 2023, atmospheric methane (CH 4 ) saw a decrease in the annual growth rate following record increases from 2020 to 2022. Recent changes in CH 4 remain difficult to quantify due to delays in near‐real‐time (NRT) carbon cycle estimates. However, NRT models and subseasonal‐to‐seasonal (S2S) forecasting can provide the opportunity to analyze ongoing climate events, leading to a deeper understanding of the methane cycle. We applied the Lund‐Potsdam‐Jena Earth‐Observation‐SIMulator (LPJ‐EOSIM) to investigate methane emissions in 2023, focusing on a short‐term case study using S2S forecasts to capture wetland methane dynamics during the 2023 El Niño‐related drought in Amazonia. We modeled a 2.64 ± 5.73 Tg CH 4 decrease in global wetland methane emissions from 2022 to 2023 using an ensemble of four climate driver data sets, corresponding to ∼0.95 ppb (2.77 ppb/Tg CH 4 ), accounting for 29% of the −3.33 ppb change in atmospheric methane annual growth rate. In 2023, LPJ‐EOSIM showed tropical wetland (90°S–30°N) emissions decreased by 4.47 ± 4.79 Tg CH 4 , with Amazonia accounting for 68% of this reduction (3.05 ± 2.40 Tg CH 4 ). Meanwhile, emissions across northern latitudes (>30°N) increased by 1.83 ± 1.21 Tg CH 4 , partially offsetting the large tropical decrease. We show that the NRT LPJ‐EOSIM coupled with S2S forecasts could help forecast these anomalies in Amazonia, with reliable regional lead times at 3–5 months when forecasts were initialized following the wet‐to‐dry season transition. NRT products and S2S forecasts can provide early warning systems for improved strategic wetland CH 4 monitoring to assess impacts of climate anomalies on the carbon cycle and reduce the gap in the global methane budget.

Figure S6 shows prognostic significance of MARCH2 levels in different cancer types

Figure S8 shows effect of MARCH2 shRNA expression on growth of MDA-MB231 cells

Rationale and Objectives: To evaluate moral injury (MI) among interventional radiologists using validated assessment tools. Materials and Methods: An anonymous 29-question online survey was distributed to interventional radiologists using the Society of Interventional Radiology Connect Open Forum website, Twitter, Facebook, LinkedIn, and electronic mail. The survey consisted of demographic and practice environment questions, a global quality of life (QoL) scale (scored 1-100), the MI Symptom Scale-Healthcare Professional (MISS-HP) (scored 1-100), and two open-ended questions. A MISS-HP score >= 36 was indicative of experiencing MI. P < .05 was considered statistically significant for all two-sided tests. Results: Beginning on March 30, 2023, 365 surveys were completed over 5 days. Of the respondents, 299 (81.9%) were male, 65 (17.8%) were female, and one preferred not to disclose gender. The respondents included practicing interventional radiologists (299; 81.9%) and interventional radiologists-in-training (66; 18.1%). Practice settings included academic (146; 40.0%), community (121; 33.2%), hybrid (84; 23.0%), or other (14; 3.8%) centers. Mean QoL was 71.1 +/- 17.0 (range: 0-100) suggestive of "good" QoL. Mean QoL in the MI subgroup was significantly different from that for the rest of the group (67.6 +/- 17.0 vs. 76.6 +/- 16.0; P < 0.05). 223 (61.1%) respondents scored >= 36 on the MISS-HP, and thus were categorized as having profession-related MI. Mean MISS-HP was 39.9 +/- 12.6 (range: 10-83). Mean MISS-HP in the MI subgroup was significantly different from that for the rest of the group (47.4 +/- 9.6 vs. 28.0 +/- 5.7; P < 0.05). There was a negative correlation between MI and QoL (r = -0.4; P < 0.001). Most common themes for greatest contribution to MI were ineffective leadership, barriers to patient care, corporatization of medicine, non-physician administration, performing futile procedures, turf battles, and reduced resources. Most common themes for ways to reduce MI were more autonomy, less bureaucracy, more administrative support, physician-directed leadership, adequate staffing, changes to the medical system, physician unionization, transparency with insurance companies, more time off, and leaving medicine/retirement. Conclusion: MI is prevalent among interventional radiologists, and it negatively correlates with QoL. Future work should investigate causative factors and mitigating solutions.

PTK6 inhibition increases ubiquitination and degradation of SNAIL in TNBC. A, PTK6 inhibitor (P21d) treatment or PTK6 shRNA downregulates SNAIL in TNBC cells. Human MDA-MB-231 or mouse MMTV-myc cells were treated with P21d for 24 hours and SNAIL expression was assessed by Western blot analysis. Lysates of MDA-MB-231 cells expressing vector control or either of two independent PTK6 shRNA vectors were assessed for SNAIL expression. B, MDA-MB-231 cells overexpressing HA-ubiquitin and Flag-SNAIL were treated with P21d for 8 hours with MG132 cotreatment for the last 4 hours. Lysates were immunoprecipitated with anti-FLAG antibody and probed with anti-HA or FLAG antibody. The result is representative of three independent experiments. C, Representative images of Duolink PLA using MDA-MB-231 cells coexpressing HA-ubiquitin and Flag-SNAIL. Cells were treated with P21d (5 µmol/L) for 24 hours with MG132 cotreatment for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and FLAG (SNAIL) for PLA (red) and Phalloidin for F-actin and counterstaining. PLA signals (red)/cell were quantitated using ImageJ (left). Scale bar, 15 µm. D, Representative images of PLA using MDA-MB-231 cells overexpressing HA-ubiquitin only. Cells were treated with P21d for 24 hours and with MG132 cotreatment for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and SNAIL for PLA (red) and Phalloidin for F-actin counterstaining. PLA signals/cell were quantitated using ImageJ (left). Scale bar, 15 µm. E, Representative images of PLA using MDA-MB-231 cells overexpressing HA-ubiquitin only that were transfected with control or PTK6 siRNA for 48 hours. Cells were treated with MG132 for the last 4 hours and incubated with antibodies against HA (Ubiquitin) and SNAIL for PLA (red) and Phalloidin for F-actin counterstaining. PLA signals/cell were quantitated using Image (left). Scale bar, 15 µm. All the PLA signals were counted by ImageJ with a fixed setting. Data represent mean ± SEM. *, P = 0.05; ***, P = 0.0001 by Student t test.

Swellable microneedles (MNs) expand to mechanically interlock with wet biological tissue, offering improved adhesion and enhanced drug delivery over non-swellable counterparts. This study numerically evaluates how the material and geometric parameters of swellable MN arrays influence shape change. Using finite element simulation, MNs were subjected to unconstrained swelling, approximated via a thermal-strain analogy. Optimal MN design must support mechanical interlocking to prevent dislodgement. We observed that wet in vivo environments induce unwanted swelling-mediated curvature, hindering contact and interlocking. We quantified this bending and calibrated gel material swellability using experimental data. To counteract curling, we introduced a design approach to shift the direction of the unwanted curling and improve MN array conformability.

Figure S1 shows Kaplan-Meier curves associated with EMT driver genes

MARCH2 promotes ubiquitination and downregulation of SNAIL. A, HA-tagged WT, but not RING domain–mutant (W97A), MARCH2 downregulates SNAIL levels in MDA-MB-231 cells. B, MARCH2 coprecipitates with SNAIL in MDA-MB-231 cells coexpressing FLAG-SNAIL and HA-MARCH2. C, WT, but not RING domain– or transmembrane domain–mutant, MARCH2 ubiquitinates SNAIL. FLAG-Snail was immunoprecipitated from MDA-MB-231 cells coexpressing FLAG-Snail, HA-Ubiquitin and GST-tagged MARCH2 constructs. Immunoprecipitates were blotted with antibodies to HA to assess ubiquitination. D, MARCH2 ubiquitinates SNAIL in in vitro ubiquitination assay. Reactions were performed with recombinant SNAIL (Myc-tagged) in the presence of ubiquitin, recombinant E1 (UBE1), recombinant E2 (UBE2D3) and/or recombinant MARCH2.

Expression of SNAIL and PTK6 in TNBC. A, Prognostic significance of SNAIL expression in TNBC. KM survival curves of SNAIL expression in patients with TNBC (defined as estrogen receptor, progesterone receptor, and HER2 negative) using the METBRIC dataset (n = 290) and KM plotter (n = 734). SNAIL expression was divided along the median into low versus high expressors. B, Representative images of IHC staining for PTK6 and SNAIL expression in TNBC tissue microarray (n = 48). Low (0, 1+) and high (2+, 3+) expression was scored by two independent reviewers. C, Expression of PTK6 and SNAIL in triple-negative PDX tumors.

Abstract Epithelial–mesenchymal transition (EMT) in cancer promotes metastasis and chemotherapy resistance. A subset of triple-negative breast cancer (TNBC) exhibits a mesenchymal gene signature that is associated with poor patient outcomes. We previously identified PTK6 tyrosine kinase as an oncogenic driver of EMT in a subset of TNBC. PTK6 induces EMT by stabilizing SNAIL, a key EMT-initiating transcriptional factor. Inhibition of PTK6 activity reverses mesenchymal features of TNBC cells and suppresses their metastases by promoting SNAIL degradation via a novel mechanism. In the current study, we identify membrane-associated RING-CH2 (MARCH2) as a novel PTK6-regulated E3 ligase that promotes the ubiquitination and degradation of SNAIL protein. The MARCH2 RING domain is critical for SNAIL ubiquitination and subsequent degradation. PTK6 inhibition promotes the interaction of MARCH2 with SNAIL. Overexpression of MARCH2 exhibits tumor suppressive properties and phenocopies the effects of SNAIL downregulation and PTK6 inhibition in TNBC cells, such as inhibition of migration, anoikis resistance, and metastasis. Consistent with this, higher levels of MARCH2 expression in breast and other cancers are associated with better prognosis. We have identified MARCH2 as a novel SNAIL E3 ligase that regulates EMT and metastases of mesenchymal TNBC. Significance: EMT is a process directly linked to drug resistance and metastasis of cancer cells. We identified MARCH2 as a novel regulator of SNAIL, a key EMT driver, that promotes SNAIL ubiquitination and degradation in TNBC cells. MARCH2 is oncogene regulated and inhibits growth and metastasis of TNBC. These insights could contribute to novel strategies to therapeutically target TNBC.

PTK6 inhibition enhances MARCH2:SNAIL association. A, MDA-MB-231 cells expressing FLAG-Snail and HA-MARCH2 (WT) were treated with vehicle control or P21d for 4 or 18 hours in the presence of MG132. MARCH2–SNAIL interaction was assessed by immunoblotting FLAG immunoprecipitates with anti-HA antibody. B, Endogenous MARCH2–SNAIL interaction following P21d treatment of MDA-MB-231 was assessed by Duolink PLA. Cells were treated with DMSO or P21d for 8 hours and MG132 cotreatment for the last 4 hours. Cells were stained with antibodies to Snail and MARCH2. PLA signals/cell were quantitated using ImageJ (left). Scale bar, 15 µm.

MARCH2 suppresses growth, migration, survival, and metastases of TNBC. A, MARCH2 expression is associated with favorable prognosis in patients with breast cancer using TCGA data (n = 1,052) and METABRIC data (n = 1302). Breast cancer samples were divided into high versus low expression groups based on whether their MARCH2 expression level was higher than upper quartile or lower than lower quartile. B, WT, but not RING domain–mutant (W97A), MARCH2 inhibits growth and invasive branching of MDA-MB-231 cells in 3D Matrigel cultures. Representative phase contrast images of 3D cultures of MDA-MB-231 cells overexpressing vector control, WT or RING domain–mutant (W97A) MARCH2 are shown. Scale bars, 30 µm. C, Cell Titer-glo 3D was used to measure cell viability of MDA-MB-231 cells expressing control, WT or RING domain–mutant MARCH2 in 3D cultures. Data present mean ± SD. *, P = 0.05 by Student t test. D, Transwell migration assays of MDA-MB-231 cells overexpressing pLv vector control, WT or W97A MARCH2 mutant were counted by ImageJ. Data represent mean ± SEM. **, P = 0.01 by Student t test. E, Anoikis of MDA-MB-231 cells expressing vector control, WT or W97A mutant MARCH2 cultured in suspension for 48 hours was assessed by Annexin/PI staining and flow cytometry. F, Coexpression of SNAIL restores migratory capacity of MARCH2-expressing MDA-MB-231 cells. MDA-MB-231 cells coexpressing GFP-MARCH2 and vector control or SNAIL were generated and assessed in transwell assays. Data are representative of three independent experiments each performed with technical triplicates. Data represent mean ± SD. ***, P < 0.001 by Student t test. G, SNAIL restores invasive branching and growth of MARCH2-overexpressing MDA-MB-231 cells in 3D Matrigel cultures. Cell viability was assessed using 3D Cell Titer Glo assays. Data present mean ± SD. ***, P < 0.001 by Student t test.

Figure S5 shows effect of MARCH2 on levels of EMT driver proteins

MARCH2 is a novel PTK6-regulated SNAIL E3 ligase. A, Schema of siRNA screening strategy used to identify PTK6-regulated SNAIL E3 ligases. B, MARCH2 siRNA (SMART Pool) identified as top candidate siRNA that restores SNAIL expression in P21d-treated MDA-MB-231/SNAIL- Luciferase cells by Western analysis. C, MARCH2 siRNA (SMART POOL or individual) prevents P21d-induced SNAIL downregulation in MDA-MB-231 cells.

Figure S3 shows validation of Proximity Ligation Assay to detect protein ubiquitination