Phase 2 study of neoadjuvant enzalutamide and paclitaxel for luminal androgen receptor-enriched TNBC: Trial results and insights into "ARness".

Luminal androgen receptor (LAR)-enriched triple-negative breast cancer (TNBC) is a distinct subtype. The efficacy of AR inhibitors and the relevant biomarkers in neoadjuvant therapy (NAT) are yet to be determined. We tested the combination of the AR inhibitor enzalutamide (120 mg daily by mouth) and paclitaxel (80 mg/m2 weekly intravenously) (ZT) for 12 weeks as NAT for LAR-enriched TNBC. Eligibility criteria included a percentage of cells expressing nuclear AR by immunohistochemistry (iAR) of at least 10% and a reduction in sonographic volume of less than 70% after four cycles of doxorubicin and cyclophosphamide. Twenty-four patients were enrolled. Ten achieved a pathologic complete response or residual cancer burden-I. ZT was safe, with no unexpected side effects. An iAR of at least 70% had a positive predictive value of 0.92 and a negative predictive value of 0.97 in predicting LAR-enriched TNBC according to RNA-based assays. Our data support future trials of AR blockade in early-stage LAR-enriched TNBC.

Outcomes of older adults with early-stage triple-negative breast cancer (TNBC) receiving chemotherapy: a single-institution experience.

BACKGROUND: Despite lower chemotherapy use in older triple-negative breast cancer (TNBC) patients, their outcomes match younger counterparts. We compared outcomes in early-stage TNBC patients by age receiving chemotherapy at a major cancer center with a national TNBC database. METHODS: Retrospective study using institutional data on stage I-III TNBC (ER/PR < 10%) women with neoadjuvant/adjuvant chemotherapy. Based on their ages at diagnosis, patients were stratified into four categories: ≤40, 41-59, 60-69, and ≥ 70 years. Demographic and clinical characteristics recorded included race, disease stage, ER/PR positivity, treatment regimen, lymphatic or vascular invasion (LVI), histologic grade, Ki-67 level, body mass index (BMI), and pathologic complete response (pCR) following neoadjuvant treatment and are summarized using descriptive statistics. The primary endpoints were overall survival (OS), disease-free survival (DFS), and distant disease-free survival (DDFS); all were estimated using the Kaplan-Meier method. Both univariate and multivariate (MV) Cox regressions were applied to evaluate the impact of important covariates on these time-to-event endpoints. RESULTS: Of the 2336 patients studied, 492 (21.1%) were ≤ 40 years old, 1239 (53.1%) were 41-59, 461 (19.7%) were 60-69, and 144 (6.2%) were ≥ 70. In the univariate regression model of OS/DFS/DDFS, age ≥ 70 was significantly associated with worse OS (p = 0.0217); other factors associated with worse OS were non-anthracycline-based chemotherapy, higher tumor stage, and neoadjuvant chemotherapy. The multivariate Cox regression model, adjusted for race and stage, showed no significant effects of age on OS; however, patients ≥ 70 years old who received non-anthracycline treatment combinations had worse DFS (hazard ratio = 0.349 vs. 1.049, p = 0.0293) and DDFS (hazard ratio = 0.317 vs. 1.016, p = 0.0251) than patients ≤ 40 years old. DFS from MV model after adjusting for age, race, and disease stage, the hazard ratio between anthracycline + taxane treatments and anthracycline + other treatments in patients ≥ 70 years old was statistically significantly lower than in patients ≤ 40 years old (hazard ratios [HRs] = 0.349 vs. 1.049, p = 0.0293). CONCLUSIONS: Our findings indicate that outcomes such as DFS are less favorable in older compared to younger patients with early-stage TNBC, primarily in those who did not receive an anthracycline based chemotherapy regimen.

Differential cellular stiffness across tissues that contribute to Xenopus neural tube closure.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

Maintenance Pembrolizumab Therapy in Patients with Metastatic HER2-negative Breast Cancer with Prior Response to Chemotherapy.

PURPOSE: Accumulating toxicities hinder indefinite chemotherapy for many patients with metastatic/recurrent HER2-negative breast cancer. We conducted a phase II trial of pembrolizumab monotherapy following induction chemotherapy to determine the efficacy of maintenance immunotherapy in patients with metastatic HER2-negative inflammatory breast cancer (IBC) and non-IBC triple-negative breast cancer (TNBC) and a biomarker study. PATIENTS AND METHODS: Patients with a complete response, partial response, or stable disease (SD) after at least three cycles of chemotherapy for HER2-negative breast cancer received pembrolizumab, regardless of programmed death-ligand 1 expression. Pembrolizumab (200 mg) was administered every 3 weeks until disease progression, intolerable toxicity, or 2 years of pembrolizumab exposure. The endpoints included the 4-month disease control rate (DCR), progression-free survival (PFS), overall survival, and response biomarkers in the blood. RESULTS: Of 43 treated patients, 11 had metastatic IBC and 32 non-IBC TNBC. The 4-month DCR was 58.1% [95% confidence interval (CI), 43.4-72.9]. For all patients, the median PFS was 4.8 months (95% CI, 3.0-7.1 months). The toxicity profile was similar to the previous pembrolizumab monotherapy study. Patients with high T-cell clonality at baseline had a longer PFS with pembrolizumab treatment than did those with low T-cell clonality (10.4 vs. 3.6 months, P = 0.04). Patients who achieved SD also demonstrated a significant increase in T-cell clonality during therapy compared with those who did not achieve SD (20% vs. 5.9% mean increase, respectively; P = 0.04). CONCLUSIONS: Pembrolizumab monotherapy achieved durable treatment responses. Patients with a high baseline T-cell clonality had prolonged disease control with pembrolizumab.

Comprehensive Analysis Identifies Variability in PI3K Pathway Alterations in Triple-Negative Breast Cancer Subtypes.

PURPOSE: The PI3K pathway is frequently altered in triple-negative breast cancer (TNBC). Limited cell line and human data suggest that TNBC tumors characterized as mesenchymal (M) and luminal androgen receptor (LAR) subtypes have increased incidence of alterations in the PI3K pathway. The impact of PI3K pathway alterations across TNBC subtypes is poorly understood. METHODS: Pretreatment tumor was evaluated from operable TNBC patients enrolled on a clinical trial of neoadjuvant therapy (NAT; A Robust TNBC Evaluation fraMework to Improve Survival [ClinicalTrials.gov identifier: NCT02276443]). Tumors were characterized into seven TNBC subtypes per Pietenpol criteria (basal-like 1, basal-like 2, immunomodulatory, M, mesenchymal stem-like, LAR, and unstable). Using whole-exome sequencing, RNA sequencing, and immunohistochemistry for PTEN, alterations were identified in 32 genes known to activate the PI3K pathway. Alterations in each subtype were associated with pathologic response to NAT. RESULTS: In evaluated patients (N = 177), there was a significant difference in the incidence of PI3K pathway alterations across TNBC subtypes (P < .01). The highest incidence of alterations was seen in LAR (81%), BL2 (79%), and M (62%) subtypes. The odds ratio for pathologic complete response (pCR) in the presence of PIK3CA mutation, PTEN mutation, and/or PTEN loss was highest in the LAR subtype and lowest in the M subtype, but these findings did not reach statistical significance. Presence of PIK3CA mutation was associated with pCR in the LAR subtype (P = .02). CONCLUSION: PI3K pathway alteration can affect response to NAT in TNBC, and targeted agents may improve outcomes, particularly in patients with M and LAR TNBC.

TBCRC 039: a phase II study of preoperative ruxolitinib with or without paclitaxel for triple-negative inflammatory breast cancer.

BACKGROUND: Patients with inflammatory breast cancer (IBC) have overall poor clinical outcomes, with triple-negative IBC (TN-IBC) being associated with the worst survival, warranting the investigation of novel therapies. Preclinical studies implied that ruxolitinib (RUX), a JAK1/2 inhibitor, may be an effective therapy for TN-IBC. METHODS: We conducted a randomized phase II study with nested window-of-opportunity in TN-IBC. Treatment-naïve patients received a 7-day run-in of RUX alone or RUX plus paclitaxel (PAC). After the run-in, those who received RUX alone proceeded to neoadjuvant therapy with either RUX + PAC or PAC alone for 12 weeks; those who had received RUX + PAC continued treatment for 12 weeks. All patients subsequently received 4 cycles of doxorubicin plus cyclophosphamide prior to surgery. Research tumor biopsies were performed at baseline (pre-run-in) and after run-in therapy. Tumors were evaluated for phosphorylated STAT3 (pSTAT3) by immunostaining, and a subset was also analyzed by RNA-seq. The primary endpoint was the percent of pSTAT3-positive pre-run-in tumors that became pSTAT3-negative. Secondary endpoints included pathologic complete response (pCR). RESULTS: Overall, 23 patients were enrolled, of whom 21 completed preoperative therapy. Two patients achieved pCR (8.7%). pSTAT3 and IL-6/JAK/STAT3 signaling decreased in post-run-in biopsies of RUX-treated samples, while sustained treatment with RUX + PAC upregulated IL-6/JAK/STAT3 signaling compared to RUX alone. Both treatments decreased GZMB+ T cells implying immune suppression. RUX alone effectively inhibited JAK/STAT3 signaling but its combination with PAC led to incomplete inhibition. The immune suppressive effects of RUX alone and in combination may negate its growth inhibitory effects on cancer cells. CONCLUSION: In summary, the use of RUX in TN-IBC was associated with a decrease in pSTAT3 levels despite lack of clinical benefit. Cancer cell-specific-targeting of JAK2/STAT3 or combinations with immunotherapy may be required for further evaluation of JAK2/STAT3 signaling as a cancer therapeutic target. TRIAL REGISTRATION: www. CLINICALTRIALS: gov , NCT02876302. Registered 23 August 2016.

Inflammatory Breast Cancer: Understanding the Patient Experience.

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive, locally advanced cancer with a 5-year survival rate of approximately 40%. Although patients with IBC likely experience significant and variable symptom burden from diagnosis through survivorship, the description of the symptom burden in this population is limited. OBJECTIVES: The purpose of this study was to describe the experience of patients with IBC and define the content domain for a patient-reported outcome measure of IBC symptom burden. METHODS: Twenty patients with IBC described their experience in single qualitative interviews. Content analysis was used to define the symptom burden content domain. Relevance ratings by a panel of experts reduced the number of items for a preliminary patient-reported outcome symptom burden measure. RESULTS: The mean (SD) participant age was 52.8 (12.0) years; 50.0% had distant metastatic disease, and 85.0% were currently receiving treatment. Content analysis revealed 45 symptoms, with 20 symptoms reported by greater than or equal to 20% of participants. All participants described localized disease-related symptoms. Treatment-related symptoms varied among participants based on the modalities received. CONCLUSION: Patients with IBC experience symptom burden that is distinct from the symptom burden experienced by patients with non-IBC. IMPLICATIONS FOR PRACTICE: Differentiating the disease-related symptoms of IBC may assist clinicians in making timely and accurate diagnoses for IBC. A disease- and treatment-specific measure of the symptom burden of IBC should be incorporated in clinical practice to allow for regular assessment and evaluation of symptom burden and implementation of evidence-based interventions for symptom management.

Clinical outcomes after 1 versus 2-3 lines of neoadjuvant therapy in stage III inflammatory breast cancer.

PURPOSE: Many stage III inflammatory breast cancer (IBC) patients experience a sufficient response to first-line (1L) neoadjuvant chemotherapy (NAC) to allow surgery, while some require additional NAC. We evaluated the pathologic complete response (pCR), breast cancer-free survival (BCFS) and overall survival (OS) among patients requiring 1 vs. 2-3 lines (L) of NAC prior to surgery. METHODS: Stage III IBC patients from 2 institutions who received 1L or 2-3L of NAC prior to surgery were identified. Hormone receptor and HER2 status, grade, and pCR were evaluated. BCFS and OS were evaluated by the Kaplan-Meier method. Multivariable Cox models were utilized to estimate the hazard ratio (HR). RESULTS: 808 eligible patients (1997-2020) were identified (median age 51 years, median follow-up 69 months). 733 (91%) had 1L and 75 (9%) had 2-3L of NAC. Grade III, triple-negative and HER2-positive disease were more prevalent in 2-3L patients. 178 (24%) 1L and 14 (19%) 2-3L patients had pCR. 376 1L patients and 41 2-3L patients had recurrences. The 5-year BCFS was worse for the 2-3L group (33 vs. 46%, HR = 1.37; 95% CI 0.99-1.91). However, in 192 patients with a pCR, BCFS was similar (76 vs. 83% in 1L vs. 2-3L, respectively). There were 308 deaths (276 among 1L and 32 among 2-3L patients). The 5-year OS in 1L vs. 2-3L was 60 vs. 53% (HR = 1.32, 95% CI 0.91-1.93). CONCLUSIONS: Among stage III IBC patients, pCR rates were similar, irrespective of the NAC lines number, and BCFS and OS were comparable with pCR after 1L and 2-3L.

Reassessing Estrogen Receptor Expression Thresholds for Breast Cancer Prognosis in HER2-negative Patients Using Shape Restricted Modeling.

Purpose: To assess the dynamic link between continuous estrogen receptor (ER) expression and long-term clinical outcomes in non-metastatic breast cancer and to identify the ideal cutoff value for ER expression to optimize endocrine therapy use. Methods: The study included 3055 female patients with stage II or III HER2-negative breast cancer. The primary outcomes were time to recurrence or death (TTR) and overall survival (OS). We used a novel shape-restricted Cox model to determine the desirable ER expression cutoff to predict breast cancer prognoses. Our novel model allows ER as a continuous variable, utilizing a flexible monotone-shaped Cox regression to assess its association with survival outcomes holistically. Results: The shape-restricted Cox model identified 10% ER as the preferred cutoff to predict TTR. The finding was confirmed by the log-rank test and standard Cox model that patients with ER ≥ 10% had TTR benefit over ER < 10% (log-rank p < 0.001). No OS or TTR benefit of adjuvant endocrine therapy was observed in patients with 1% ≤ ER < 10% (HR 0.877, 95% CI 0.481-1.600, p = 0.668 for TTR and HR 0.698, 95% CI 0.337-1.446, p = 0.333 for OS). Conclusions: Using the shape-restricted Cox model, this study suggests a potential preferred threshold of 10% for predicting TTR. The findings could assist physicians in effectively weighing the benefits and risks of adjuvant endocrine therapy for patients with ER < 10% disease, particularly in cases involving severe adverse events. Further prospective studies are warranted to validate the recommended cutoff value.

Analytical and clinical validation of PATHWAY Anti-HER-2/neu (4B5) antibody to assess HER2-low status for trastuzumab deruxtecan treatment in breast cancer.

In DESTINY-Breast04 (DB-04), safety and efficacy of HER2-targeted antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd) in previously treated HER2-low unresectable/metastatic breast cancer were established. This manuscript describes the analytical validation of PATHWAY Anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody (PATHWAY HER2 (4B5)) to assess HER2-low status and its clinical performance in DB-04. Preanalytical processing and tissue staining parameters were evaluated to determine their impact on HER2 scoring. The recommended antibody staining procedure provided the optimal tumor staining, and deviations in cell conditioning and/or antibody incubation times resulted in unacceptable negative control staining and/or HER2-low status changes. Comparisons between antibody lots, kit lots, instruments, and day-to-day runs showed overall percent agreements (OPAs) exceeding 97.9%. Inter-laboratory reproducibility showed OPAs of ≥97.4% for all study endpoints. PATHWAY HER2 (4B5) was utilized in DB-04 for patient selection using 1340 tumor samples (59.0% metastatic, 40.7% primary, (0.3% missing data); 74.3% biopsy, 25.7% resection/excisions). Overall, 77.6% (823/1060) of samples were HER2-low by both central and local testing, with the level of concordance differing by sample region of origin and collection date. In DB-04, the efficacy of T-DXd over chemotherapy of physician's choice was consistent, regardless of the characteristics of the sample used (primary or metastatic, archival, or newly collected, biopsy or excision/resection). These results demonstrate that PATHWAY HER2 (4B5) is precise and reproducible for scoring HER2-low status and can be used with multiple breast cancer sample types for reliably identifying patients whose tumors have HER2-low expression and are likely to derive clinical benefit from T-DXd.

PTEN in triple-negative breast carcinoma: protein expression and genomic alteration in pretreatment and posttreatment specimens.

Background: Recent advances have been made in targeting the phosphoinositide 3-kinase pathway in breast cancer. Phosphatase and tensin homolog (PTEN) is a key component of that pathway. Objective: To understand the changes in PTEN expression over the course of the disease in patients with triple-negative breast cancer (TNBC) and whether PTEN copy number variation (CNV) by next-generation sequencing (NGS) can serve as an alternative to immunohistochemistry (IHC) to identify PTEN loss. Methods: We compared PTEN expression by IHC between pretreatment tumors and residual tumors in the breast and lymph nodes after neoadjuvant chemotherapy in 96 patients enrolled in a TNBC clinical trial. A correlative analysis between PTEN protein expression and PTEN CNV by NGS was also performed. Results: With a stringent cutoff for PTEN IHC scoring, PTEN expression was discordant between pretreatment and posttreatment primary tumors in 5% of patients (n = 96) and between posttreatment primary tumors and lymph node metastases in 9% (n = 33). A less stringent cutoff yielded similar discordance rates. Intratumoral heterogeneity for PTEN loss was observed in 7% of the patients. Among pretreatment tumors, PTEN copy numbers by whole exome sequencing (n = 72) were significantly higher in the PTEN-positive tumors by IHC compared with the IHC PTEN-loss tumors (p < 0.0001). However, PTEN-positive and PTEN-loss tumors by IHC overlapped in copy numbers: 14 of 60 PTEN-positive samples showed decreased copy numbers in the range of those of the PTEN-loss tumors. Conclusion: Testing various specimens by IHC may generate different PTEN results in a small proportion of patients with TNBC; therefore, the decision of testing one versus multiple specimens in a clinical trial should be defined in the patient inclusion criteria. Although a distinct cutoff by which CNV differentiated PTEN-positive tumors from those with PTEN loss was not identified, higher copy number of PTEN may confer positive PTEN, whereas lower copy number of PTEN would necessitate additional testing by IHC to assess PTEN loss. Trial registration: NCT02276443.

Inflammatory breast cancer biomarker identification by simultaneous TGIRT-seq profiling of coding and non-coding RNAs in tumors and blood.

Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype, but lags in biomarker identification. Here, we used an improved Thermostable Group II Intron Reverse Transcriptase RNA sequencing (TGIRT-seq) method to simultaneously profile coding and non-coding RNAs from tumors, PBMCs, and plasma of IBC and non-IBC patients and healthy donors. Besides RNAs from known IBC-relevant genes, we identified hundreds of other overexpressed coding and non-coding RNAs (p≤0.001) in IBC tumors and PBMCs, including higher proportions with elevated intron-exon depth ratios (IDRs), likely reflecting enhanced transcription resulting in accumulation of intronic RNAs. As a consequence, differentially represented protein-coding gene RNAs in IBC plasma were largely intron RNA fragments, whereas those in healthy donor and non-IBC plasma were largely fragmented mRNAs. Potential IBC biomarkers in plasma included T-cell receptor pre-mRNA fragments traced to IBC tumors and PBMCs; intron RNA fragments correlated with high IDR genes; and LINE-1 and other retroelement RNAs that we found globally up-regulated in IBC and preferentially enriched in plasma. Our findings provide new insights into IBC and demonstrate advantages of broadly analyzing transcriptomes for biomarker identification. The RNA-seq and data analysis methods developed for this study may be broadly applicable to other diseases.

The immunogram of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most aggressive and fatal clinical presentation of breast cancer. Despite the term "inflammatory", based on the clinical presentation, IBC is biologically driven by an immunosuppressive tumor microenvironment (TME). Whether IBC can be switched into an immune-inflamed TME by immune-checkpoint inhibitors (ICIs) is a matter of debate. Presently, measurable biomarkers of IBC-TME have never been synthetized into a comprehensive portray of the immune-milieu (i.e., an immunogram), describing the immune-vulnerability of IBC and potentially predicting the response to ICIs. We propose an immunogram for IBC, based on preclinical and clinical studies, including six parameters: the presence of immune-effector cells, of immune-suppressive cells and of immune checkpoints, the general immune status, the activation of immune-suppressive pathways, the tumor foreignness. The IBC immunogram suggests the existence of a preexisting immune TME that is suppressed by mechanisms of immune-escape but might be restored by ICIs. The combination of chemotherapy and ICIs in patients with IBC is based on a strong biological rationale. However, the design and the development of clinical trials assessing the incorporation of ICIs raise many methodological and practical issues. In parallel with the further comprehension of IBC biology, the prospective validation and integration of biomarkers predictive of response to ICIs are warranted.

Predictive Roles of Baseline Stromal Tumor-Infiltrating Lymphocytes and Ki-67 in Pathologic Complete Response in an Early-Stage Triple-Negative Breast Cancer Prospective Trial.

High stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved pathologic complete response (pCR) in triple-negative breast cancer (TNBC). We hypothesize that integrating high sTILs and additional clinicopathologic features associated with pCR could enhance our ability to predict the group of patients on whom treatment de-escalation strategies could be tested. In this prospective early-stage TNBC neoadjuvant chemotherapy study, pretreatment biopsies from 408 patients were evaluated for their clinical and demographic features, as well as biomarkers including sTILs, Ki-67, PD-L1 and androgen receptor. Multivariate logistic regression models were developed to generate a computed response score to predict pCR. The pCR rate for the entire cohort was 41%. Recursive partitioning analysis identified ≥20% as the optimal cutoff for sTILs to denote 35% (143/408) of patients as having high sTILs, with a pCR rate of 59%, and 65% (265/408) of patients as having low sTILs, with a pCR rate of 31%. High Ki-67 (cutoff > 35%) was identified as the only predictor of pCR in addition to sTILs in the training set. This finding was verified in the testing set, where the highest computed response score encompassing both high sTILa and high Ki-67 predicted a pCR rate of 65%. Integrating Ki67 and sTIL may refine the selection of early stage TNBC patients for neoadjuvant clinical trials evaluating de-escalation strategies.

Intestinal expression patterns of transcription factors and markers for interstitial cells in the larval zebrafish.

For the digestion of food, it is important for the gut to be differentiated regionally and to have proper motor control. However, the number of transcription factors that regulate its development is still limited. Meanwhile, the interstitial cells of the gastrointestinal (GI) tract are necessary for intestinal motility in addition to the enteric nervous system. There are anoctamine1 (Ano1)-positive and platelet-derived growth factor receptor α (Pdgfra)-positive interstitial cells in mammal, but Pdgfra-positive cells have not been reported in the zebrafish. To identify new transcription factors involved in GI tract development, we used RNA sequencing comparing between larval and adult gut. We isolated 40 transcription factors that were more highly expressed in the larval gut. We demonstrated expression patterns of the 13 genes, 7 of which were newly found to be expressed in the zebrafish larval gut. Six of the 13 genes encode nuclear receptors. The osr2 is expressed in the anterior part, while foxP4 in its distal part. Also, we reported the expression pattern of pdgfra for the first time in the larval zebrafish gut. Our data provide fundamental knowledge for studying vertebrate gut regionalization and motility by live imaging using zebrafish.

Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.