Overcoming osimertinib resistance with AKT inhibition in EGFRm-driven Non-Small-Cell-Lung-Cancer with PIK3CA/PTEN alterations.

PURPOSE: Osimertinib is an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) indicated for the treatment of EGFR mutated (EGFRm)-driven lung adenocarcinomas. Osimertinib significantly improves progression-free survival in first-line treated patients with EGFRm advanced NSCLC. Despite the durable disease control, the majority of patients receiving osimertinib eventually develop disease progression. EXPERIMENTAL DESIGN: ctDNA profiling analysis on-progression plasma samples from patients treated with osimertinib in both first (Phase 3, FLAURA trial) and second-line trials (Phase 3, AURA3 trial) revealed a high prevalence of PIK3CA/AKT/PTEN alterations. In vitro and in vivo evidence using CRISPR engineered NSCLC cell lines and PXD models support a functional role for PIK3CA and PTEN mutations in the development of osimertinib resistance. RESULTS: These alterations are functionally relevant as EGFRm NSCLC cells with engineered PIK3CA/AKT/PTEN alterations develop resistance to osimertinib and can be re-sensitized by treatment with the combination of osimertinib and the AKT inhibitor capivasertib. Moreover, xenograft and PDX in vivo models with PIK3CA/AKT/PTEN alterations display limited sensitivity to osimertinib relative to models without alteration, and in these double mutant models capivasertib and osimertinib combination elicits an improved anti-tumor effect versus osimertinib alone. CONCLUSIONS: Together, this approach offers a potential treatment strategy for patients with EGFRm-driven NSCLC that have a sub-optimal response, or develop resistance, to osimertinib through PIK3CA/AKT/PTEN alterations.

Accelerating Drug Development Using Spatial Multi-omics.

SUMMARY: Spatial biology approaches enabled by innovations in imaging biomarker platforms and artificial intelligence-enabled data integration and analysis provide an assessment of patient and disease heterogeneity at ever-increasing resolution. The utility of spatial biology data in accelerating drug programs, however, requires balancing exploratory discovery investigations against scalable and clinically applicable spatial biomarker analysis.

Histopathological biomarkers for predicting the tumour accumulation of nanomedicines.

The clinical prospects of cancer nanomedicines depend on effective patient stratification. Here we report the identification of predictive biomarkers of the accumulation of nanomedicines in tumour tissue. By using supervised machine learning on data of the accumulation of nanomedicines in tumour models in mice, we identified the densities of blood vessels and of tumour-associated macrophages as key predictive features. On the basis of these two features, we derived a biomarker score correlating with the concentration of liposomal doxorubicin in tumours and validated it in three syngeneic tumour models in immunocompetent mice and in four cell-line-derived and six patient-derived tumour xenografts in mice. The score effectively discriminated tumours according to the accumulation of nanomedicines (high versus low), with an area under the receiver operating characteristic curve of 0.91. Histopathological assessment of 30 tumour specimens from patients and of 28 corresponding primary tumour biopsies confirmed the score's effectiveness in predicting the tumour accumulation of liposomal doxorubicin. Biomarkers of the tumour accumulation of nanomedicines may aid the stratification of patients in clinical trials of cancer nanomedicines.

Large-scale Pan-cancer Cell Line Screening Identifies Actionable and Effective Drug Combinations.

Oncology drug combinations can improve therapeutic responses and increase treatment options for patients. The number of possible combinations is vast and responses can be context-specific. Systematic screens can identify clinically relevant, actionable combinations in defined patient subtypes. We present data for 109 anticancer drug combinations from AstraZeneca's oncology small molecule portfolio screened in 755 pan-cancer cell lines. Combinations were screened in a 7 × 7 concentration matrix, with more than 4 million measurements of sensitivity, producing an exceptionally data-rich resource. We implement a new approach using combination Emax (viability effect) and highest single agent (HSA) to assess combination benefit. We designed a clinical translatability workflow to identify combinations with clearly defined patient populations, rationale for tolerability based on tumor type and combination-specific "emergent" biomarkers, and exposures relevant to clinical doses. We describe three actionable combinations in defined cancer types, confirmed in vitro and in vivo, with a focus on hematologic cancers and apoptotic targets. SIGNIFICANCE: We present the largest cancer drug combination screen published to date with 7 × 7 concentration response matrices for 109 combinations in more than 750 cell lines, complemented by multi-omics predictors of response and identification of "emergent" combination biomarkers. We prioritize hits to optimize clinical translatability, and experimentally validate novel combination hypotheses. This article is featured in Selected Articles from This Issue, p. 695.

Biomarker-directed targeted therapy plus durvalumab in advanced non-small-cell lung cancer: a phase 2 umbrella trial.

For patients with non-small-cell lung cancer (NSCLC) tumors without currently targetable molecular alterations, standard-of-care treatment is immunotherapy with anti-PD-(L)1 checkpoint inhibitors, alone or with platinum-doublet therapy. However, not all patients derive durable benefit and resistance to immune checkpoint blockade is common. Understanding mechanisms of resistance-which can include defects in DNA damage response and repair pathways, alterations or functional mutations in STK11/LKB1, alterations in antigen-presentation pathways, and immunosuppressive cellular subsets within the tumor microenvironment-and developing effective therapies to overcome them, remains an unmet need. Here the phase 2 umbrella HUDSON study evaluated rational combination regimens for advanced NSCLC following failure of anti-PD-(L)1-containing immunotherapy and platinum-doublet therapy. A total of 268 patients received durvalumab (anti-PD-L1 monoclonal antibody)-ceralasertib (ATR kinase inhibitor), durvalumab-olaparib (PARP inhibitor), durvalumab-danvatirsen (STAT3 antisense oligonucleotide) or durvalumab-oleclumab (anti-CD73 monoclonal antibody). Greatest clinical benefit was observed with durvalumab-ceralasertib; objective response rate (primary outcome) was 13.9% (11/79) versus 2.6% (5/189) with other regimens, pooled, median progression-free survival (secondary outcome) was 5.8 (80% confidence interval 4.6-7.4) versus 2.7 (1.8-2.8) months, and median overall survival (secondary outcome) was 17.4 (14.1-20.3) versus 9.4 (7.5-10.6) months. Benefit with durvalumab-ceralasertib was consistent across known immunotherapy-refractory subgroups. In ATM-altered patients hypothesized to harbor vulnerability to ATR inhibition, objective response rate was 26.1% (6/23) and median progression-free survival/median overall survival were 8.4/22.8 months. Durvalumab-ceralasertib safety/tolerability profile was manageable. Biomarker analyses suggested that anti-PD-L1/ATR inhibition induced immune changes that reinvigorated antitumor immunity. Durvalumab-ceralasertib is under further investigation in immunotherapy-refractory NSCLC.ClinicalTrials.gov identifier: NCT03334617.

Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer.

BACKGROUND/OBJECTIVE: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. METHODS: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. RESULTS: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3ß, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3ß as a GSK3ß inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. CONCLUSION: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3ß.

Multiomic profiling of breast cancer cells uncovers stress MAPK-associated sensitivity to AKT degradation.

More than 50% of human tumors display hyperactivation of the serine/threonine kinase AKT. Despite evidence of clinical efficacy, the therapeutic window of the current generation of AKT inhibitors could be improved. Here, we report the development of a second-generation AKT degrader, INY-05-040, which outperformed catalytic AKT inhibition with respect to cellular suppression of AKT-dependent phenotypes in breast cancer cell lines. A growth inhibition screen with 288 cancer cell lines confirmed that INY-05-040 had a substantially higher potency than our first-generation AKT degrader (INY-03-041), with both compounds outperforming catalytic AKT inhibition by GDC-0068. Using multiomic profiling and causal network integration in breast cancer cells, we demonstrated that the enhanced efficacy of INY-05-040 was associated with sustained suppression of AKT signaling, which was followed by induction of the stress mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK). Further integration of growth inhibition assays with publicly available transcriptomic, proteomic, and reverse phase protein array (RPPA) measurements established low basal JNK signaling as a biomarker for breast cancer sensitivity to AKT degradation. Together, our study presents a framework for mapping the network-wide signaling effects of therapeutically relevant compounds and identifies INY-05-040 as a potent pharmacological suppressor of AKT signaling.

Imaging tumor lactate is feasible for identifying intermediate-risk prostate cancer patients with postsurgical biochemical recurrence.

While radical prostatectomy remains the mainstay of prostate cancer (PCa) treatment, 20 to 40% of patients develop postsurgical biochemical recurrence (BCR). A particularly challenging clinical cohort includes patients with intermediate-risk disease whose risk stratification would benefit from advanced approaches that complement standard-of-care diagnostic tools. Here, we show that imaging tumor lactate using hyperpolarized 13C MRI and spatial metabolomics identifies BCR-positive patients in two prospective intermediate-risk surgical cohorts. Supported by spatially resolved tissue analysis of established glycolytic biomarkers, this study provides the rationale for multicenter trials of tumor metabolic imaging as an auxiliary tool to support PCa treatment decision-making.

The Next-Generation Oral Selective Estrogen Receptor Degrader Camizestrant (AZD9833) Suppresses ER+ Breast Cancer Growth and Overcomes Endocrine and CDK4/6 Inhibitor Resistance.

Oral selective estrogen receptor degraders (SERD) could become the backbone of endocrine therapy (ET) for estrogen receptor-positive (ER+) breast cancer, as they achieve greater inhibition of ER-driven cancers than current ETs and overcome key resistance mechanisms. In this study, we evaluated the preclinical pharmacology and efficacy of the next-generation oral SERD camizestrant (AZD9833) and assessed ER-co-targeting strategies by combining camizestrant with CDK4/6 inhibitors (CDK4/6i) and PI3K/AKT/mTOR-targeted therapy in models of progression on CDK4/6i and/or ET. Camizestrant demonstrated robust and selective ER degradation, modulated ER-regulated gene expression, and induced complete ER antagonism and significant antiproliferation activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) breast cancer cell lines and patient-derived xenograft (PDX) models. Camizestrant also delivered strong antitumor activity in fulvestrant-resistant ESR1wt and ESR1m PDX models. Evaluation of camizestrant in combination with CDK4/6i (palbociclib or abemaciclib) in CDK4/6-naive and -resistant models, as well as in combination with PI3Kαi (alpelisib), mTORi (everolimus), or AKTi (capivasertib), indicated that camizestrant was active with CDK4/6i or PI3K/AKT/mTORi and that antitumor activity was further increased by the triple combination. The response was observed independently of PI3K pathway mutation status. Overall, camizestrant shows strong and broad antitumor activity in ER+ breast cancer as a monotherapy and when combined with CDK4/6i and PI3K/AKT/mTORi. SIGNIFICANCE: Camizestrant, a next-generation oral SERD, shows promise in preclinical models of ER+ breast cancer alone and in combination with CDK4/6 and PI3K/AKT/mTOR inhibitors to address endocrine resistance, a current barrier to treatment.

Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC.

Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models.

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB- T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB- resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Pharmacokinetics of the Akt Serine/Threonine Protein Kinase Inhibitor, Capivasertib, Administered to Healthy Volunteers in the Presence and Absence of the CYP3A4 Inhibitor Itraconazole.

Capivasertib is a potent, selective inhibitor of all 3 Akt isoforms (Akt1/2/3), and it is currently being tested in Phase III trials for the treatment of prostate and breast cancer. To investigate the effect of a cytochrome P450 3A4 (CYP3A4) inhibitor on the pharmacokinetics of capivasertib, a Phase I drug-drug interaction study of capivasertib and itraconazole was conducted in 11 healthy volunteers (median age, 54 years). The 8-day study had 3 stages: Participants received a single dose of capivasertib 80 mg in Stage 1, 4 doses of itraconazole 200 mg over 3 days in Stage 2, and a final dose of capivasertib 80 mg coadministered with itraconazole 200 mg in Stage 3. Capivasertib pharmacokinetics were examined in Stages 1 and 3. Itraconazole coadministration increased the maximum plasma concentration of capivasertib and total capivasertib exposure (area under the concentration-time curve from time of administration to infinity) by 1.70-fold (90% confidence interval, 1.56-1.86) and 1.95-fold (90% confidence interval, 1.82-2.10), respectively.

Perfusion Air Culture of Precision-Cut Tumor Slices: An Ex Vivo System to Evaluate Individual Drug Response under Controlled Culture Conditions.

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

Therapeutic targeting of tumour myeloid cells.

Myeloid cells are pivotal within the immunosuppressive tumour microenvironment. The accumulation of tumour-modified myeloid cells derived from monocytes or neutrophils - termed 'myeloid-derived suppressor cells' - and tumour-associated macrophages is associated with poor outcome and resistance to treatments such as chemotherapy and immune checkpoint inhibitors. Unfortunately, there has been little success in large-scale clinical trials of myeloid cell modulators, and only a few distinct strategies have been used to target suppressive myeloid cells clinically so far. Preclinical and translational studies have now elucidated specific functions for different myeloid cell subpopulations within the tumour microenvironment, revealing context-specific roles of different myeloid cell populations in disease progression and influencing response to therapy. To improve the success of myeloid cell-targeted therapies, it will be important to target tumour types and patient subsets in which myeloid cells represent the dominant driver of therapy resistance, as well as to determine the most efficacious treatment regimens and combination partners. This Review discusses what we can learn from work with the first generation of myeloid modulators and highlights recent developments in modelling context-specific roles for different myeloid cell subtypes, which can ultimately inform how to drive more successful clinical trials.

mTOR inhibition amplifies the anti-lymphoma effect of PI3Kß/δ blockage in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kß/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kß/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kß/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kß/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kß/δ signaling and thus identifies a previously unappreciated role of the PI3Kß isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kß/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.