Plasma ctDNA liquid biopsy of IDH1, TERTp, and EGFRvIII mutations in glioma.

Background: Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene mutations IDH1, TERTp, and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy. Methods: We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient). DNA was isolated from plasma and analyzed for IDH1, TERTp, and EGFRvIII mutations using ddPCR. Results: Total cfDNA was significantly associated with tumor grade, tumor volume, and both overall and progression-free survival for all gliomas as well as the grade 4 glioblastoma subgroup, but was not reliably associated with changes in tumor volume/progression during the patients' postoperative time course. IDH1 mutation was detected with 84% overall sensitivity across all plasma samples and 77% in the preoperative samples alone; however, IDH1 mutation plasma levels were not associated with tumor progression or survival. IDH1m plasma levels were not associated with pre- or postsurgery progression or survival. The TERTp C228T mutation was detected in the plasma ctDNA in 88% but the C250T variant in only 49% of samples. The EGFRvIII mutation was detected in plasma in 5 out of 7 patients (71%) with tissue EGFRvIII mutations in tumor tissue. Conclusions: Plasma ctDNA mutations detected with ddPCR provide excellent diagnostic sensitivity for IDH1, TERTp-C228T, and EGFRvIII mutations in glioma patients. Total cfDNA may also assist with prognostic information. Further studies are needed to validate these findings and the clinical role of ctDNA in glioma.

Plasma ctDNA enables early detection of temozolomide resistance mutations in glioma.

Background: Liquid biopsy based on circulating tumor DNA (ctDNA) is a novel tool in clinical oncology, however, its use has been limited in glioma to date, due to low levels of ctDNA. In this study, we aimed to demonstrate that sequencing techniques optimized for liquid biopsy in glioma patients can detect ctDNA in plasma with high sensitivity and with potential clinical utility. Methods: We investigated 10 glioma patients with tumor tissue available from at least 2 surgical operations, who had 49 longitudinally collected plasma samples available for analysis. Plasma samples were sequenced with CAPP-seq (AVENIO) and tissue samples with TSO500. Results: Glioma-derived ctDNA mutations were detected in 93.8% of plasma samples. 25% of all mutations detected were observed in plasma only. Mutations of the mismatch repair (MMR) genes MSH2 and MSH6 were the most frequent circulating gene alterations seen after temozolomide treatment and were frequently observed to appear in plasma prior to their appearance in tumor tissue at the time of surgery for recurrence. Conclusions: This pilot study suggests that plasma ctDNA in glioma is feasible and may provide sensitive and complementary information to tissue biopsy. Furthermore, plasma ctDNA detection of new MMR gene mutations not present in the initial tissue biopsy may provide an early indication of the development of chemotherapy resistance. Additional clinical validation in larger cohorts is needed.

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma; DIPG), are uniformly fatal brain tumors that lack effective treatment. Analysis of CRISPR/Cas9 loss-of-function gene deletion screens identified PIK3CA and MTOR as targetable molecular dependencies across patient derived models of DIPG, highlighting the therapeutic potential of the blood-brain barrier-penetrant PI3K/Akt/mTOR inhibitor, paxalisib. At the human-equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic glucose feedback and increased insulin levels commensurate with patients using PI3K inhibitors. To exploit genetic dependence and overcome resistance while maintaining compliance and therapeutic benefit, we combined paxalisib with the antihyperglycemic drug metformin. Metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending survival of orthotopic models. DIPG models treated with paxalisib increased calcium-activated PKC signaling. The brain penetrant PKC inhibitor enzastaurin, in combination with paxalisib, synergistically extended the survival of multiple orthotopic patient-derived and immunocompetent syngeneic allograft models; benefits potentiated in combination with metformin and standard-of-care radiotherapy. Therapeutic adaptation was assessed using spatial transcriptomics and ATAC-Seq, identifying changes in myelination and tumor immune microenvironment crosstalk. Collectively, this study has identified what we believe to be a clinically relevant DIPG therapeutic combinational strategy.

A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

Spatial architecture of high-grade glioma reveals tumor heterogeneity within distinct domains.

Background: High-grade gliomas (HGGs) are aggressive primary brain cancers with poor response to standard regimens, driven by immense heterogeneity. In isocitrate dehydrogenase (IDH) wild-type HGG (glioblastoma, GBM), increased intratumoral heterogeneity is associated with more aggressive disease. Methods: Spatial technologies can dissect complex heterogeneity within the tumor ecosystem by preserving cellular organization in situ. We employed GeoMx digital spatial profiling, CosMx spatial molecular imaging, Xenium in situ mapping and Visium spatial gene expression in experimental and validation patient cohorts to interrogate the transcriptional landscape in HGG. Results: Here, we construct a high-resolution molecular map of heterogeneity in GBM and IDH-mutant patient samples to investigate the cellular communities that compose HGG. We uncovered striking diversity in the tumor landscape and degree of spatial heterogeneity within the cellular composition of the tumors. The immune distribution was diverse between samples, however, consistently correlated spatially with distinct tumor cell phenotypes, validated across tumor cohorts. Reconstructing the tumor architecture revealed two distinct niches, one composed of tumor cells that most closely resemble normal glial cells, associated with microglia, and the other niche populated by monocytes and mesenchymal tumor cells. Conclusions: This primary study reveals high levels of intratumoral heterogeneity in HGGs, associated with a diverse immune landscape within spatially localized regions.

Matrix Selection for the Visualization of Small Molecules and Lipids in Brain Tumors Using Untargeted MALDI-TOF Mass Spectrometry Imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging allows for the study of metabolic activity in the tumor microenvironment of brain cancers. The detectable metabolites within these tumors are contingent upon the choice of matrix, deposition technique, and polarity setting. In this study, we compared the performance of three different matrices, two deposition techniques, and the use of positive and negative polarity in two different brain cancer types and across two species. Optimal combinations were confirmed by a comparative analysis of lipid and small-molecule abundance by using liquid chromatography-mass spectrometry and RNA sequencing to assess differential metabolites and enzymes between normal and tumor regions. Our findings indicate that in the tumor-bearing brain, the recrystallized α-cyano-4-hydroxycinnamic acid matrix with positive polarity offered superior performance for both detected metabolites and consistency with other techniques. Beyond these implications for brain cancer, our work establishes a workflow to identify optimal matrices for spatial metabolomics studies.

Tumor cell-derived spermidine promotes a pro-tumorigenic immune microenvironment in glioblastoma via CD8+ T cell inhibition.

The glioblastoma microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine is elevated in the glioblastoma tumor microenvironment. Exogenous administration of spermidine drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and phenotype. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+T cell number and function.

Discovery of a highly potent, selective, orally bioavailable inhibitor of KAT6A/B histone acetyltransferases with efficacy against KAT6A-high ER+ breast cancer.

KAT6A, and its paralog KAT6B, are histone lysine acetyltransferases (HAT) that acetylate histone H3K23 and exert an oncogenic role in several tumor types including breast cancer where KAT6A is frequently amplified/overexpressed. However, pharmacologic targeting of KAT6A to achieve therapeutic benefit has been a challenge. Here we describe identification of a highly potent, selective, and orally bioavailable KAT6A/KAT6B inhibitor CTx-648 (PF-9363), derived from a benzisoxazole series, which demonstrates anti-tumor activity in correlation with H3K23Ac inhibition in KAT6A over-expressing breast cancer. Transcriptional and epigenetic profiling studies show reduced RNA Pol II binding and downregulation of genes involved in estrogen signaling, cell cycle, Myc and stem cell pathways associated with CTx-648 anti-tumor activity in ER-positive (ER+) breast cancer. CTx-648 treatment leads to potent tumor growth inhibition in ER+ breast cancer in vivo models, including models refractory to endocrine therapy, highlighting the potential for targeting KAT6A in ER+ breast cancer.

Correlating MRI features with additional genetic markers and patient survival in histological grade 2-3 IDH-mutant astrocytomas.

PURPOSE: The increasing importance of molecular markers for classification and prognostication of diffuse gliomas has prompted the use of imaging features to predict genotype ("radiogenomics"). CDKN2A/B homozygous deletion has only recently been added to the diagnostic paradigm for IDH[isocitrate dehydrogenase]-mutant astrocytomas; thus, associated radiogenomic literature is sparse. There is also little data on whether different IDH mutations are associated with different imaging appearances. Furthermore, given that molecular status is now generally obtained routinely, the additional prognostic value of radiogenomic features is less clear. This study correlated MRI features with CDKN2A/B status, IDH mutation type and survival in histological grade 2-3 IDH-mutant brain astrocytomas. METHODS: Fifty-eight grade 2-3 IDH-mutant astrocytomas were identified, 50 with CDKN2A/B results. IDH mutations were stratified into IDH1-R132H and non-canonical mutations. Background and survival data were obtained. Two neuroradiologists independently assessed the following MRI features: T2-FLAIR mismatch (<25%, 25-50%, >50%), well-defined tumour margins, contrast-enhancement (absent, wispy, solid) and central necrosis. RESULTS: 8/50 tumours with CDKN2A/B results demonstrated homozygous deletion; slightly shorter survival was not significant (p=0.571). IDH1-R132H mutations were present in 50/58 (86%). No MRI features correlated with CDKN2A/B status or IDH mutation type. T2-FLAIR mismatch did not predict survival (p=0.977), but well-defined margins predicted longer survival (HR 0.36, p=0.008), while solid enhancement predicted shorter survival (HR 3.86, p=0.004). Both correlations remained significant on multivariate analysis. CONCLUSION: MRI features did not predict CDKN2A/B homozygous deletion, but provided additional positive and negative prognostic information which correlated more strongly with prognosis than CDKN2A/B status in our cohort.

Flow Cytometry Identification of Cell Compartments in the Murine Brain.

Glioma can be modelled in the murine brain through the induction of genetically engineered mouse models or intracranial transplantation. Gliomas (oligodendroglioma and astrocytoma) are thought to arise from neuronal and glial progenitor populations in the brain and are poorly infiltrated by immune cells. An improved understanding of oligodendrocytes, astrocytes, and the immune environment throughout tumor development will enhance the analysis and development of brain cancer models. Here, we describe the isolation and analysis of murine brain cell types using a combination of flow cytometry and quantitative RT-PCR strategies to analyze these individual cell populations in vivo.

BRAF-mediated brain tumors in adults and children: A review and the Australian and New Zealand experience.

The mitogen-activated protein kinase (MAPK) pathway signaling pathway is one of the most commonly mutated pathways in human cancers. In particular, BRAF alterations result in constitutive activation of the rapidly accelerating fibrosarcoma-extracellular signal-regulated kinase-MAPK significant pathway, leading to cellular proliferation, survival, and dedifferentiation. The role of BRAF mutations in oncogenesis and tumorigenesis has spurred the development of targeted agents, which have been successful in treating many adult cancers. Despite advances in other cancer types, the morbidity and survival outcomes of patients with glioma have remained relatively stagnant. Recently, there has been recognition that MAPK dysregulation is almost universally present in paediatric and adult gliomas. These findings, accompanying broad molecular characterization of gliomas, has aided prognostication and offered opportunities for clinical trials testing targeted agents. The use of targeted therapies in this disease represents a paradigm shift, although the biochemical complexities has resulted in unexpected challenges in the development of effective BRAF inhibitors. Despite these challenges, there are promising data to support the use of BRAF inhibitors alone and in combination with MEK inhibitors for patients with both low-grade and high-grade glioma across age groups. Safety and efficacy data demonstrate that many of the toxicities of these targeted agents are tolerable while offering objective responses. Newer clinical trials will examine the use of these therapies in the upfront setting. Appropriate duration of therapy and durability of response remains unclear in the glioma patient cohort. Longitudinal efficacy and toxicity data are needed. Furthermore, access to these medications remains challenging outside of clinical trials in Australia and New Zealand. Compassionate access is limited, and advocacy for mechanism of action-based drug approval is ongoing.

Moderately hypofractionated versus conventionally fractionated radiation therapy with temozolomide for young and fit patients with glioblastoma: an institutional experience and meta-analysis of literature.

PURPOSE: Shorter hypofractionated radiation therapy (HF-RT) schedules may have radiobiological, patient convenience and healthcare resource advantages over conventionally fractionated radiation therapy (CF-RT) in glioblastoma (GBM). We report outcomes of young, fit GBM patients treated with HF-RT and CF-RT during the COVID-19 pandemic, and a meta-analysis of HF-RT literature in this patient subgroup. METHODS: Hospital records of patients with IDH-wildtype GBM treated with HF-RT (50 Gy/20 fractions) and CF-RT (60 Gy/30 fractions) between January 2020 and September 2021 were reviewed. Overall survival (OS) and progression-free survival (PFS) were estimated using the Kaplan-Meier method. Univariable analysis was performed using Cox regression analysis. A systematic search and meta-analysis of studies from January 2000 to January 2022 was performed. RESULTS: 41 patients were treated (HF-RT:15, CF-RT:26). For both HF-RT and CF-RT groups, median age was 58 years and 80-90% were ECOG 0-1. There were more methylated tumours in the HF-RT group. All patients received concurrent/adjuvant temozolomide. At 19.2 months median follow-up, median OS was 19.8 months and not-reached for HF-RT and CF-RT (p = 0.5), and median PFS was 7.7 and 5.8 months, respectively (p = 0.8). HF-RT or CF-RT did not influence OS/PFS on univariable analysis. Grade 3 radionecrosis rate was 6.7% and 7.7%, respectively. 15 of 1135 studies screened from a systematic search were eligible for meta-analysis. For studies involving temozolomide, pooled median OS and PFS with HF-RT were 17.5 and 9.9 months (927 and 862 patients). Studies using shortened HF-RT schedules reported 0-2% Grade 3 radionecrosis rates. CONCLUSION: HF-RT may offer equivalent outcomes and reduce treatment burden compared to CF-RT in young, fit GBM patients.

How has the COVID-19 pandemic impacted clinical care and research in Neuro-Oncology?

The COVID-19 pandemic has challenged the continued delivery of healthcare globally. Due to disease risk, clinicians were forced to re-evaluate the safety and priorities of pre-pandemic care. Neuro-oncology presents unique challenges, as patients can deteriorate rapidly without intervention. These challenges were also observed in countries with reduced COVID-19 burden with centres required to rapidly develop strategies to maintain efficient and equitable care. This review aims to summarise the impact of the pandemic on clinical care and research within the practice of Neuro-oncology. A narrative review of the literature was performed using MEDLINE and EMBASS and results screened using PRISMA guidelines with relevant inclusion and exclusion criteria. Search strategies included variations of 'Neuro-oncology' combined with COVID-19 and other clinical-related terms. Most adult and paediatric neurosurgical centres experienced reductions in new referrals and operations for brain malignancies, and those who did present for treatment frequently had operations cancelled or delayed. Many radiation therapy and medical oncology centres altered treatment plans to mitigate COVID-19 risk for patients and staff. New protocols were developed that aimed to reduce in-person visits and reduce the risk of developing severe complications from COVID-19. The COVID-19 pandemic has presented many challenges to the provision of safe and accessible healthcare. Despite these challenges, some benefits to healthcare provision such as the use of telemedicine are likely to remain in future practice. Neuro-oncology staff must remain vigilant to ensure patient and staff safety.

Adult medulloblastoma in an Australian population.

Medulloblastoma in adult patients is a rare condition with limited contemporary demographic and treatment outcome data available in an Australian population. We conducted a retrospective review of patterns of care and outcomes of adult patients diagnosed with medulloblastoma treated at major neuro-oncology centres across Australia between January 2010 and December 2019. A total of 80 patients were identified and the median follow-up after diagnosis was 59.2 (range 0.5-204) months. A variety of chemotherapy regimens were used in the adjuvant and recurrent settings. The median overall survival (mOS) was 78 months (IQR 17.5-94.8). Patients who had no residual disease post-resection or with SHH-subtype tumours had a numerically longer 5-year survival rate than those with residual disease post resection or non-SHH subtypes respectively. The median time to recurrence from diagnosis was 18.4 months. The median OS from 1st relapse was 22.1 months (95% CI 11.7-31.4) and mOS from second relapse was 10.2 months (95% CI 6.6 - NR). This is the largest dataset examining patterns of care of adult patients with medulloblastoma in an Australian population. Substantial variation existed in the chemotherapy agents used in the adjuvant and recurrent setting. As has been demonstrated in a paediatric population, trials such as the upcoming EORTC 1634-BTG/NOA-23 trial (PersoMed-1 study) which are tailoring treatments to molecular profiles are likely to improve outcome in adult medulloblastoma.

PALVEN: phase Ib trial of palbociclib, letrozole and venetoclax in estrogen receptor- and BCL2-positive advanced breast cancer.

The addition of a CDK4/6 inhibitor to endocrine therapy improves progression-free and overall survival in women with metastatic estrogen receptor-positive breast cancer. In that setting, CDK4/6 inhibitors induce a potent cell-cycle arrest (which may be accompanied by tumor senescence) but fail to induce apoptotic cell death. Venetoclax is a potent inhibitor of BCL2, a pro-survival protein overexpressed in the majority of estrogen receptor-positive cancers. Pre-clinical findings indicate that venetoclax augments tumor response to the CDK4/6 inhibitor palbociclib by triggering apoptosis, including in senescent cells. The PALVEN phase Ib trial will further examine this finding. The primary objective is to identify the maximum tolerated dose and determine the recommended phase II dose for palbociclib, letrozole and venetoclax combination therapy. Clinical Trial Registration: NCT03900884 (ClinicalTrials.gov).

The current 'gold standard' treatment for estrogen receptor-positive, HER2-negative metastatic breast cancer is endocrine therapy with a CDK4/6 inhibitor. This combination improves tumor response and patient outcomes, primarily by reducing tumor cell growth. Paradoxically, less killing of tumor cells is observed in the presence of a CDK4/6 inhibitor. The authors hypothesize that co-treatment with venetoclax, an inhibitor of the BCL2 survival protein, will help trigger tumor death, thereby further improving tumor responses and patient outcomes. As a first step, combination therapy comprising letrozole, palbociclib and venetoclax will be tested in a phase I trial to identify the recommended doses for subsequent studies.

In vivo genome-editing screen identifies tumor suppressor genes that cooperate with Trp53 loss during mammary tumorigenesis.

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53+/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis.

Perioperative clinical trials for glioma: Raising the bar.

Gliomas are a heterogeneous group of primary brain cancers with poor survival despite multimodality therapy that includes surgery, radiation and chemotherapy. Numerous clinical trials have investigated systemic therapies in glioma, but have largely been negative. Multiple factors have contributed to the lack of progress including tumour heterogeneity, the tumour micro-environment and presence of the blood-brain barrier, as well as extrinsic factors relating to trial design, such as the lack of a contemporaneous biopsy at the time of treatment. A number of strategies have been proposed to progress new agents into the clinic. Here, we review the progress of perioperative, including phase 0 and 'window of opportunity', studies and provide recommendations for trial design in the development of new agents for glioma. The incorporation of pre- and post-treatment biopsies in glioma early phase trials will provide valuable pharmacokinetic and pharmacodynamic data and also determine the target or biomarker effect, which will guide further development of new agents. Perioperative 'window of opportunity' studies must use drugs with a recommended-phase-2-dose, known safety profile and adequate blood-brain barrier penetration. Drugs shown to have on-target effects in perioperative trials can then be evaluated further in a larger cohort of patients in an adaptive trial to increase the efficiency of drug development.

A phase 1 trial of 4-(N-(S-penicillaminylacetyl)amino)-phenylarsonous acid (PENAO) in patients with advanced solid tumours.

PURPOSE: This phase I study was conducted to evaluate the safety and Maximum Tolerated Dose of PENAO (4-(N-(S-penicillaminylacetyl)amino)-phenylarsonous acid), a second-generation organic arsenical with anti-mitochondrial activity, when given as a continuous intravenous infusion (CIVI), in patients with advanced solid tumours. METHODS: Eligibility criteria for this trial included age ≥ 18 years, advanced solid tumour, ECOG Performance Status ≤ 1 and adequate organ function. PENAO was administered by CIVI, with dose levels initially increased by infusion duration in a 21-day cycle at a fixed daily dose and then increased daily dose. Standard dose-limiting toxicity (DLT) definitions were used in a "3 + 3" design. Patients had regular monitoring of toxicity and efficacy. Pharmacokinetic assays of serum and urine As were performed. RESULTS: Twenty-six patients were treated across 8 dose levels. The only dose-limiting toxicity (DLT) observed was fatigue, that occurred in one patient treated at the highest dose level of 9 mg/m2/day. No significant organ toxicity or objective responses were observed, although there were two patients with stable disease lasting up to 7 months. Pharmacokinetic analysis unexpectedly indicated a half-life of 9-19 days, invalidating the CIVI dosing resulting in discontinuation of the study before the RP2D was defined. CONCLUSIONS: PENAO was administered by CIVI at dose levels up to 9 mg/m2/day with only one DLT noted. Pharmacokinetic studies invalidated the rationale for continuous dosing and led to discontinuation of the trial without defining a RP2D. Future clinical development of PENAO will use intermittent dosing schedule, alone and in combination with rapamycin.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant.

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.