Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

Probing the optical properties and toxicological profile of zinc tungstate nanorods.

Zinc tungstate is a semiconductor known for its favorable photocatalytic, photoluminescence, and scintillation properties, coupled with its relatively low cost, reduced toxicity, and high stability in biological and catalytic environments. In particular, zinc tungstate evinces scintillation properties, namely the ability to emit visible light upon absorption of energetic radiation such as x rays, which has led to applications not only as radiation detectors but also for biomedical applications involving the delivery of optical light to deep tissue, such as photodynamic therapy and optogenetics. Here, we report on the synthesis of zinc tungstate nanorods generated via an optimized but facile method, which allows for synthetic control over the aspect ratio of the as-synthesized anisotropic motifs via rational variation of the solution pH. We investigate the effect of aspect ratio on their resulting photoluminescent and radioluminescent properties. We further demonstrate the potential of these zinc tungstate nanorods for biomedical applications, such as photodynamic therapy for cancer treatment, by analyzing their toxicological profile within cell lines and neurons.

The evolution of RET inhibitor resistance in RET-driven lung and thyroid cancers.

The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression.

TRK xDFG Mutations Trigger a Sensitivity Switch from Type I to II Kinase Inhibitors.

On-target resistance to next-generation TRK inhibitors in TRK fusion-positive cancers is largely uncharacterized. In patients with these tumors, we found that TRK xDFG mutations confer resistance to type I next-generation TRK inhibitors designed to maintain potency against several kinase domain mutations. Computational modeling and biochemical assays showed that TRKAG667 and TRKCG696 xDFG substitutions reduce drug binding by generating steric hindrance. Concurrently, these mutations stabilize the inactive (DFG-out) conformations of the kinases, thus sensitizing these kinases to type II TRK inhibitors. Consistently, type II inhibitors impede the growth and TRK-mediated signaling of xDFG-mutant isogenic and patient-derived models. Collectively, these data demonstrate that adaptive conformational resistance can be abrogated by shifting kinase engagement modes. Given the prior identification of paralogous xDFG resistance mutations in other oncogene-addicted cancers, these findings provide insights into rational type II drug design by leveraging inhibitor class affinity switching to address recalcitrant resistant alterations. SIGNIFICANCE: In TRK fusion-positive cancers, TRK xDFG substitutions represent a shared liability for type I TRK inhibitors. In contrast, they represent a potential biomarker of type II TRK inhibitor activity. As all currently available type II agents are multikinase inhibitors, rational drug design should focus on selective type II inhibitor creation.This article is highlighted in the In This Issue feature, p. 1.

Fluoxetine effects on behavior and adult hippocampal neurogenesis in female C57BL/6J mice across the estrous cycle.

RATIONALE: Some mood disorders, such as major depressive disorder, are more prevalent in women than in men. However, historically preclinical studies in rodents have a lower inclusion rate of females than males, possibly due to the fact that behavior can be affected by the estrous cycle. Several studies have demonstrated that chronic antidepressant treatment can decrease anxiety-associated behaviors and increase adult hippocampal neurogenesis in male rodents. OBJECTIVE: Very few studies have looked at the effects of antidepressants on behavior and neurogenesis across the estrous cycle in naturally cycling female rodents. METHODS: Here, we analyze the effects of chronic treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine (Prozac) on behavior and adult hippocampal neurogenesis in naturally cycling C57BL/6J females across all four phases of the estrous cycle. RESULTS: In naturally cycling C57BL/6J females, fluoxetine decreases negative valence behaviors associated with anxiety in the elevated plus maze and novelty-suppressed feeding task, reduces immobility time in forced swim test, and increases adult hippocampal neurogenesis. Interestingly, the effects of fluoxetine on several negative valence behavior and adult hippocampal neurogenesis measures were mainly found within the estrus and diestrus phases of the estrous cycle. CONCLUSIONS: Taken together, these data are the first to illustrate the effects of fluoxetine on behavior and adult hippocampal neurogenesis across all four phases of the murine estrous cycle.

TRK Fusions Are Enriched in Cancers with Uncommon Histologies and the Absence of Canonical Driver Mutations.

PURPOSE: TRK inhibitors achieve marked tumor-agnostic efficacy in TRK fusion-positive cancers and consequently are now an established standard of care. Little is known, however, about the demographics, outcomes, response to alternative standard therapies, or genomic characteristics of TRK fusion-positive cancers. EXPERIMENTAL DESIGN: Utilizing a center-wide screening program involving more than 26,000 prospectively sequenced patients, genomic and clinical data from all cases with TRK fusions were extracted. An integrated analysis was performed of genomic, therapeutic, and phenomic outcomes. RESULTS: We identified 76 cases with confirmed TRK fusions (0.28% overall prevalence) involving 48 unique rearrangements and 17 cancer types. The presence of a TRK fusion was associated with depletion of concurrent oncogenic drivers (P < 0.001) and lower tumor mutation burden (P < 0.001), with the exception of colorectal cancer where TRK fusions cooccur with microsatellite instability (MSI-H). Longitudinal profiling in a subset of patients indicated that TRK fusions were present in all sampled timepoints in 82% (14/17) of cases. Progression-free survival on first-line therapy, excluding TRK inhibitors, administered for advanced disease was 9.6 months [95% confidence interval (CI), 4.8-13.2]. The best overall response rate achieved with chemotherapy containing-regimens across all lines of therapy was 63% (95% CI, 41-81). Among 12 patients treated with checkpoint inhibitors, a patient with MSI-H colorectal cancer had the only observed response. CONCLUSIONS: TRK fusion-positive cancers can respond to alternative standards of care, although efficacy of immunotherapy in the absence of other predictive biomarkers (MSI-H) appears limited. TRK fusions are present in tumors with simple genomes lacking in concurrent drivers that may partially explain the tumor-agnostic efficacy of TRK inhibitors.

HER2-Mediated Internalization of Cytotoxic Agents in ERBB2 Amplified or Mutant Lung Cancers.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody-drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy. SIGNIFICANCE: T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627.

PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression.

The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1 to program chromatin and ER transcriptional output.

Resistance to TRK inhibition mediated by convergent MAPK pathway activation.

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1-8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9-11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design.

Neratinib is effective in breast tumors bearing both amplification and mutation of ERBB2 (HER2).

Mutations in ERBB2, the gene encoding epidermal growth factor receptor (EGFR) family member HER2, are common in and drive the growth of "HER2-negative" (not ERBB2 amplified) tumors but are rare in "HER2-positive" (ERBB2 amplified) breast cancer. We analyzed DNA-sequencing data from HER2-positive patients and used cell lines and a patient-derived xenograft model to test the consequence of HER2 mutations on the efficacy of anti-HER2 agents such as trastuzumab, lapatinib, and neratinib, an irreversible pan-EGFR inhibitor. HER2 mutations were present in ~7% of HER2-positive tumors, all of which were metastatic but not all were previously treated. Compared to HER2 amplification alone, in both patients and cultured cell lines, the co-occurrence of HER2 mutation and amplification was associated with poor response to trastuzumab and lapatinib, the standard-of-care anti-HER2 agents. In mice, xenografts established from a patient whose HER2-positive tumor acquired a D769Y mutation in HER2 after progression on trastuzumab-based therapy were resistant to trastuzumab or lapatinib but were sensitive to neratinib. Clinical data revealed that six heavily pretreated patients with tumors bearing coincident HER2 amplification and mutation subsequently exhibited a statistically significant response to neratinib monotherapy. Thus, these findings indicate that coincident HER2 mutation reduces the efficacy of therapies commonly used to treat HER2-positive breast cancer, particularly in metastatic and previously HER2 inhibitor-treated patients, as well as potentially in patients scheduled for first-line treatment. Therefore, we propose that clinical studies testing the efficacy of neratinib are warranted selectively in breast cancer patients whose tumors carry both amplification and mutation of ERBB2/HER2.