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RTK activity plays a key role in regulating p110a degradation

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer.Significance:The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1

PDF file - 751K, Bliss Scores of A549 and H647 Across Dose Matrix. Percent inhibition relative to DMSO control was measured using CellTiterGlo. Bliss scores were calculated as described in the materials and methods

Although mitogen-activated protein (MAP)–extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non–small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. Mol Cancer Ther; 12(6); 853–64. ©2013 AACR.

PDF file - 115K, Synthetic route for G-963

PDF file - 39K, Summary of Combination Matrices for Navitoclax and G-963 in Pancreatic Cancer Cell Lines. The data reflect the average of three independent runs performed in quadruplicate

PDF file - 558K, Biochemical Selectivity of G-963. Percent inhibition of various kinases treated with 1uM G-963. Assays were performed at Invitrogen using their SelectScreenR Kinase profiling service

PDF file - 581K, Correlative Biomarker Analysis. A) Western blots illustrating baseline levels of total Bad and phospho-S-112 Bad. Correlative analysis of Bliss sums and baseline levels of B) phospho-S-112 Bad (Spearman ρ = -0.46; P = 0.02)

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Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers.Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents.Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents.Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. Clin Cancer Res; 19(7); 1760–72. ©2012 AACR.

PDF file - 954K, Supplemental Figures: Figure S1. Chemical structure of the Akt inhibitor GDC-0068. Figure S2. Effect of GDC-0068 on cell cycle and apoptotic response on PC-3 and MCF7-neo/HER2 cell lines. Figure S3. Western blot analysis of indicated proteins in isogenic MCF10A cells with and without PTEN knockout in the presence of 20 or 0.2 ng/ml EGF. Figure S4. Representative immunohistochemistry staining images of cleaved caspase-3 in TOV-21G.x1 tumors. Figure S5. Combination effects between GDC-0068 and chemotherapeutic agents in vitro.

XLSX file - 74K, Table S1. Enzymatic potency, selectivity and cellular potency of GDC-0068 Table S2. GDC-0068 IC50 profile on cell viability and genetic background of cancer cell lines Table S3. Maximum percent body weight changes of mice treated with GDC-0068 single agent and in combination with chemotherapeutic agents

Unser Mobilitätsverhalten wird durch hochautomatisch fahrenden Fahrzeuge grundlegend verändert. Automatisches Fahren wird es mitreisenden und fahrzeugführenden Personen ermöglichen, sich während der Fahrt zu beschäftigen, ohne das Verkehrsgeschehen verfolgen zu müssen. Eine solche – fahrfremde – Beschäftigung kann das Risiko für Kinetose (häufig auf Deutsch Reisekrankheit) erhöhen. Zur Entwicklung effektiver Gegenmaßnahmen bieten sich Probandenstudien an. Mithilfe dieser können unter anderem angemessene Aktivierungszeitpunkte, Anregungsdauern oder -intensitäten ermittelt werden. Für eine solche nutzernahe Entwicklung ist die objektive Erfassung der Kinetose am Menschen ein unverzichtbares Werkzeug. Es wird daher im Rahmen dieser Arbeit der Zusammenhang zwischen Kinetose im Fahrzeug und physiologischen sowie kontextualen Größen ermittelt. Die erhobenen Parameter werden in drei Clustern untersucht: physiologische Daten, die über direkten Kontakt mit dem Menschen erhoben werden (klassisch), physiologische Daten, die mit minimalem Kontakt erhoben werden (kontaktlos) sowie kontextuale Daten (kontextual). Die Erhebung der Clusterparameter greift in unterschiedlichem Maße in das Umfeld der Personen ein. Es wird eine Abhängigkeit von dem Mobilitätserlebnis und der Nutzerakzeptanz zu der Immersion der Messmethode angenommen. Die klassischen Daten umfassen Hautleitfähigkeit, Innenohrtemperatur, Atmung und Puls, während bei den kontaktlosen Daten die Gesichtstemperatur und -blässe durch Kameras sowie Temperatur und Feuchte mithilfe von Sensoren in der Sitzfläche erfasst wurden. Die kontextualen Daten wurden über Fragebögen erhoben und umfassen das Alter, das Geschlecht und eine selbst eingeschätzte Empfindlichkeit für Kinetose. Eine anwendungsnahe Untersuchung wurde mithilfe eines Fahrversuchs auf einem Testgelände durchgeführt. Reproduzierbare Stop-and-Go-Fahrten provozierten bei 40 Personen Kinetose. Die Teilnehmenden betrachteten dabei ein Video, während ihre subjektiven Kinetoseausprägungen und physiologischen Daten aufgenommen wurden. Aufgrund der Versuchsgestaltung mit wiederholtem Testen und intra- sowie interindividuellen Unterschieden, zeigten die Daten Abhängigkeiten innerhalb der Versuchsdurchläufe und der Teilnehmenden. Diese Zusammenhänge wurden in der Datenanalyse mithilfe von Multilevel-Modellen berücksichtigt. Bei der Betrachtung der einzelnen physiologischen Größen zeigen die Innenohrtemperatur, die Gesichtsblässe sowie die geschätzte Empfindlichkeit einen signifikanten positiven Zusammenhang mit der Kinetoseausprägung. Die Betrachtung der Cluster ergibt, dass Sensitivität und Präzision für die klassischen sowie kontaktlosen Parameter zwischen 40 % und 70 % liegen und die Modelle damit eine niedrige Güte haben. Dementgegen bewirkt das kontextuale Cluster ein Modell, welches mit einer Präzision von 72 % und einer Sensitivität von 98 %, eine höhere Güte hat. Parameterkombinationen des klassischen und des kontaktlosen Clusters detektieren Kinetose gut, wenn individuelle Varianzen bekannt sind. Ohne diese, sind die Modelle für eine finale Nutzung im Fahrzeug unzureichend geeignet. Einzelne Parameter der Cluster zeigen jedoch deutliche Zusammenhänge mit Kinetose. Das kontextuale Cluster hat ein höheres Potenzial, doch es erlaubt, entgegen den anderen Clustern, keine zeitechte Detektion der Kinetose, sondern ausschließlich eine Schätzung, ob Kinetose im Allgemeinen eintreten wird. Eine Verbesserung könnte in zukünftigen Arbeiten durch eine umfassendere Erfassung der Umwelt und der Nutzenden bewirkt werden. Langzeit-Aufnahmen von physiologischen Signalen und damit gute Referenzwerte der Nutzenden bieten beispielsweise Wearables. Dies kann ein weiterer Schritt zu einer objektiven Kinetosedetektion sein, die letztlich die Entwicklung von kinetosegerechter Mobilität ermöglichen kann., Highly automated vehicles will change our mobility behavior fundamentally. It will allow all passengers to engage in non-driving related tasks. This will increase the risk for motion sickness, requiring the development of effective countermeasures through user studies. These user studies can provide insight on activation time points, durations and intensities of the countermeasures, for example. Such a user-centered development requires objective detection of motion sickness. In the scope of this work the relationship between carsickness and physiological, as well as contextual parameters, is being analyzed. The extent of the invasion on the passenger is connected to the riding experience and user acceptance. The recorded parameters are split into three groups, which differ in their extent of invasion on the passenger: physiological data recorded with direct contact (classic), physiological data recorded with no or indirect contact to the passenger (contactless), and contextual data (contextual). The distinction of the three groups enables an evaluation of objective physiological parameters with the best available data recording quality (classic) in parallel to objective parameters having a lower quality, but little or no interference with the passenger (contactless and contextual). It is assumed that the mobility experience and user acceptance are depending on the immersion of the measuring method. The classic data include skin conductance, inner ear temperature, respiration, and pulse, while the contactless data include the facial temperature and pallor, as well as temperature and humidity recorded from a seat mat. The contextual data were collected using questionnaires and comprise of age, gender, and self-estimated susceptibility for motion sickness. Application-oriented research was conducted by means of a driving study on a test track. During a reproducible stop-and-go ride, forty participants had to watch a video. During that motion sickness provoking situation, physiological data and minutely subjective motion sickness were recorded. Repeated measures of the individuals as well as inter- and intraindividual differences, leading to dependencies within the trials and the participants, were studied using multilevel models. The inspection of the single parameters shows a significant positive relationship between the subjective motion sickness and the inner ear temperature, the facial pallor, and the estimated theoretical susceptibility. The models for the classic and contactless clusters have an overall moderate performance, with sensitivity and precision being between 40 % and 70 %. In contrast, the contextual model has a higher performance with a sensitivity of 98 % and precision of 72 %. Considering an in-vehicle application for a user, the classic and the contactless parameters have an overall insufficient performance of detecting motion sickness, which will not lead to the development of trust and acceptance. However, single parameters did show clear relationships to motion sickness. The contextual data showed a better potential of detecting motion sickness, but in contrast to the other clusters, would not allow a real-time detection, but rather an estimation of the overall susceptibility. Future work should consider a broader range of passenger data as well as additional environmental data. Insights of physiological data over a longer time range and good references of single users could be achieved with wearables. Additional energy is required to record physiological high-quality data with a non-intrusive method in the vehicle environment, which bears many artifacts for sensitive sensors. This thesis reveals important parameters and methodological implications for in-vehicle implementation of motion sickness detection. It is an important step towards robust objective motion sickness detection, which will eventually allow the development of motion-sickness oriented mobility.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib

The phosphatidylinositol 3kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Hotspot mutations of PIK3CA are common in all subtypes of breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent ATP-competitive inhibitor of PI3K alpha (IC50 = 0.038 ± 0.003 nM), thereby inhibiting the phosphorylation of membranebound PIP2 to PIP3. Biochemically, GDC-0077 is more than 300fold selective over the other Class I PI3K isoforms, beta, delta, and gamma, and more than 2000-fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell-based assays. Mechanism of action studies indicate that GDC-0077 treatment leads to degradation of the mutant PI3K alpha protein in a proteasome dependent fashion, without changing WT PI3K alpha levels. Drug treatment results in sustained reduction of PI3K pathway biomarkers pAkt and pPRAS40, inhibition of cell proliferation, and increased apoptosis in human PIK3CA mutant breast cancer cell lines, to a greater extent than other non-degrading inhibitors such as alpelisib. Combination studies of GDC-0077 with the CDK4/6 inhibitor palbociclib demonstrate a stronger effect in estrogen-depleted HR+ PIK3CA mutant breast cell lines, compared to cells grown in the presence of estrogen. Daily dosing of GDC-0077 in PIK3CA mutant breast cancer patient derived xenograft (PDX) models (n=4) resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo combination efficacy studies of GDC-0077 with fulvestrant and palbociclib also indicate the triple combination results in the greatest efficacy. Collectively, our preclinical data support evaluation of GDC-0077 in a triple combination with endocrine therapy and CDK4/6 inhibition in the clinic, in patients with locally advanced or metastatic hormone receptor positive breast cancer whose tumors harbor mutant PIK3CA. Citation Format: Kyle Edgar, Rebecca Hong, Kyung Song, Steven Schmidt, Marc Hafner, Alfonso Arrazate, Erin Williams, Cecile De La Cruz, Jason Oeh, Deepak Sampath, Steve Staben, Lori Friedman. GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-23.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Burns are a leading cause of morbidity and mortality worldwide. Understanding when and how burns occur, as well as the differences between countries, would aid prevention efforts. A review of burn injuries occurring between July 2009 and June 2021 was undertaken using data from the Burns Registry of Australia and New Zealand. Peak injury times were identified on a country-by-country basis. Variations in demographic and injury event profiles between countries were compared using descriptive statistics. There were 26,925 admissions recorded across the two countries (23,323 for Australia; 3602 for New Zealand). The greatest number of injuries occurred between 6 PM to 7 PM in Australia (1871, 8.0%) and between 5 PM to 6 PM in New Zealand (280, 7.8%). In both countries, scalds accounted for the greatest proportion of injuries during peak times (988, 45.8%), but a greater proportion of young children (under three years) sustained burns during New Zealand’s peak times. The number of burn injuries associated with the preparation and/or consumption of food offers an opportunity for a targeted prevention program that may yield benefits across the two countries. Age- and mechanism-related differences in the profile of burn-injured patients need to be considered when developing and implementing such a program.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. Significance: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors. See related commentary by Vanhaesebroeck et al., p. 20. This article is highlighted in the In This Issue feature, p. 1

The phosphatidylinositol 3 kinase (PI3K)/AKT/ mechanistic target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/AKT/mTOR signaling pathway through multiple different mechanisms has been described in solid tumor malignancies, including activating and transforming mutations and amplification of PIK3CA, that encodes the p110alpha subunit of PI3K. Indeed, PIK3CA hotspot mutations are highly prevalent in breast cancer, occurring in approximately 40% of HR+ tumors. The clinical candidate GDC-0077 is a potent inhibitor of PI3Kalpha (IC50 = 0.038 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is >300-fold more selective for PI3Kalpha over the other class I PI3K isoforms (beta, delta, and gamma) and >2000-fold more selective over PIK family members. Furthermore, GDC-0077 is more selective for mutant versus wild-type PI3Kalpha in cell based assays. The improved biochemical selectivity of GDC-0077 relative to PI3Kdelta translated in human CD69+ B-cells, which are primarily dependent on PI3Kdelta for proliferation and survival, and were more sensitive (based on reduction of cell number) to the PI3Kalpha/delta selective inhibitor taselisib (GDC-0032) than to GDC-0077. Mechanism of action (MOA) studies indicate that GDC-0077 selectively degrades mutant PI3Kalpha in a proteasome-dependent fashion resulting in reduction of PI3K pathway activity biomarkers such as pAKT and pPRAS40, inhibition of cell proliferation, and increased apoptosis in human PIK3CA-mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, oral daily treatment of patient-derived PIK3CA-mutant breast cancer xenograft models with GDC-0077 resulted in tumor regressions, induction of apoptosis, and a reduction of pAKT, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or a CDK4/6 inhibitor (palbociclib). Collectively, preclinical studies provide rationale for evaluating GDC-0077, a PI3Kalpha selective inhibitor that degrades mutant p110alpha protein, as a single agent and in combination with endocrine and targeted therapies that may provide additional benefit to patients with locally advanced or metastatic hormone receptor+ breast cancers that harbor PIK3CA mutations. Citation Format: Hong R, Edgar K, Song K, Steven S, Young A, Hamilton P, Arrazate A, De La Cruz C, Chan C, Pang J, Salphati L, Belvin M, Nannini M, Staben S, Friedman L, Sampath D. GDC-0077 is a selective PI3Kalpha inhibitor that demonstrates robust efficacy in PIK3CA mutant breast cancer models as a single agent and in combination with standard of care therapies [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-14.

Targeted inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) can induce regression of tumors bearing activating mutations in the Ras pathway but rarely leads to tumor eradication. Although combining MEK inhibition with T-cell-directed immunotherapy might lead to more durable efficacy, T cell responses are themselves at least partially dependent on MEK activity. We show here that MEK inhibition did profoundly block naive CD8(+) T cell priming in tumor-bearing mice, but actually increased the number of effector-phenotype antigen-specific CD8(+) T cells within the tumor. MEK inhibition protected tumor-infiltrating CD8(+) T cells from death driven by chronic TCR stimulation while sparing cytotoxic activity. Combining MEK inhibition with anti-programmed death-ligand 1 (PD-L1) resulted in synergistic and durable tumor regression even where either agent alone was only modestly effective. Thus, despite the central importance of the MAP kinase pathway in some aspects of T cell function, MEK-targeted agents can be compatible with T-cell-dependent immunotherapy.

Summary: We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target. : HPK1 is implicated in several important steps that limit T cell responsiveness, but the mechanism is poorly characterized. Hernandez et al. demonstrate that HPK1’s kinase activity is essential for attenuating T cell function. Loss of HPK1 kinase activity enhanced antitumoral responses, proving to be an attractive target for cancer immunotherapy. Keywords: HPK1, hematopoietic progenitor kinase 1, cancer immunotherapy

Expression of PD-L1, the ligand for T-cell inhibitory receptor PD-1, is one key immunosuppressive mechanism by which cancer avoids eradication by the immune system. Therapeutic use of blocking antibodies to PD-L1 or its receptor PD-1 has produced unparalleled, durable clinical responses, with highest likelihood of response seen in patients whose tumour or immune cells express PD-L1 before therapy. The significance of PD-L1 expression in each cell type has emerged as a central and controversial unknown in the clinical development of immunotherapeutics. Using genetic deletion in preclinical mouse models, here we show that PD-L1 from disparate cellular sources, including tumour cells, myeloid or other immune cells can similarly modulate the degree of cytotoxic T-cell function and activity in the tumour microenvironment. PD-L1 expression in both the host and tumour compartment contribute to immune suppression in a non-redundant fashion, suggesting that both sources could be predictive of sensitivity to therapeutic agents targeting the PD-L1/PD-1 axis., PD-L1, the ligand for T-cell inhibitory receptor PD-1, can be expressed by various cell types in the tumour microenvironment. Here, the authors show that, in mouse models, the expression of PD-L1 from both cancerous and normal host immune cells is important for suppressing anti-tumour immune responses.

The phosphoinositide 3-kinases (PI3Ks) are lipid kinases that activate the PI3K signaling pathway and play an essential role in regulating breast tumor cell growth, migration, and survival. Activating and transforming mutations in the PIK3CA gene (PI3K alpha) are commonly found in HER2+ breast cancer and ER+ breast cancer. GDC-0032 is a selective, orally bioavailable inhibitor with a Ki of 0.29 nM for PI3K alpha with 30 fold less inhibition of PI3K beta. In addition, GDC-0032 has increased single agent activity against PI3K alpha mutant and HER2 amplified cancer models in vitro and in vivo. The aim of our study was to evaluate the efficacy of GDC-0032 in breast cancer models in combination with standard of care therapeutics including taxanes, endocrine therapies, and HER2 targeted therapies. We evaluated cell viability for a range of dose concentrations of GDC-0032, as single agent and in combinations, in eleven breast cancer cell lines that harbor mutations in PI3K alpha (K111N, E545K, H1047R) and/or over-expressed HER2. GDC-0032 was combined with taxanes (paclitaxel and docetaxel), endocrine therapies (fulvestrant and tamoxifen) or anti-HER2 agents (trastuzumab, pertuzumab or ado-trastuzumab-emtansine) and synergy quantified using the Chou and Talalay method of Combination Index (C.I.). The combination of GDC-0032 with taxanes, endocrine therapies or anti-HER2 therapies were synergistic in the breast cancer cell lines tested based on C.I. values of less than 1.0. The combination of GDC-0032 with docetaxel in vitro resulted in decreased viability as a result of increased cell death. The in vitro results translated in vivo as GDC-0032 enhanced the anti-tumor activity of docetaxel and paclitaxel in MCF7 (E545K) xenografts that resulted in increased tumor regressions when GDC-0032 was dosed daily or intermittently on the same schedule as the taxane. In addition, the combination of GDC-0032 and fulvestrant or tamoxifen resulted in greater efficacy in the MCF7 xenograft model when compared to either agent alone. Enhanced efficacy was also observed when GDC-0032 was combined with trastuzumab or ado-trastuzumab-emtansine in the HER2+ BT474M1 (K111N) xenograft model. Moreover, the triple combinations of GDC-0032/trastuzumab/pertuzumab or GDC-0032/trastuzumab/docetaxel resulted in durable tumor regressions that were sustained in the HER2+ KPL-4 (H1047R) and BT474M1 xenograft models, respectively. All drug combinations with GDC-0032 were well tolerated in vivo based on minimal changes in body weight. Collectively, our preclinical data demonstrate that GDC-0032 enhances the efficacy of standard of care therapeutics in PI3K alpha mutant breast cancer models and provides a strong rationale for further evaluation in patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-02.

Purpose: We describe the preclinical pharmacology and antitumor activity of GDC-0068, a novel highly selective ATP-competitive pan-Akt inhibitor currently in clinical trials for the treatment of human cancers. Experimental Design: The effect of GDC-0068 on Akt signaling was characterized using specific biomarkers of the Akt pathway, and response to GDC-0068 was evaluated in human cancer cell lines and xenograft models with various genetic backgrounds, either as a single agent or in combination with chemotherapeutic agents. Results: GDC-0068 blocked Akt signaling both in cultured human cancer cell lines and in tumor xenograft models as evidenced by dose-dependent decrease in phosphorylation of downstream targets. Inhibition of Akt activity by GDC-0068 resulted in blockade of cell-cycle progression and reduced viability of cancer cell lines. Markers of Akt activation, including high-basal phospho-Akt levels, PTEN loss, and PIK3CA kinase domain mutations, correlate with sensitivity to GDC-0068. Isogenic PTEN knockout also sensitized MCF10A cells to GDC-0068. In multiple tumor xenograft models, oral administration of GDC-0068 resulted in antitumor activity ranging from tumor growth delay to regression. Consistent with the role of Akt in a survival pathway, GDC-0068 also enhanced antitumor activity of classic chemotherapeutic agents. Conclusions: GDC-0068 is a highly selective, orally bioavailable Akt kinase inhibitor that shows pharmacodynamic inhibition of Akt signaling and robust antitumor activity in human cancer cells in vitro and in vivo. Our preclinical data provide a strong mechanistic rationale to evaluate GDC-0068 in cancers with activated Akt signaling. Clin Cancer Res; 19(7); 1760–72. ©2012 AACR.

Summary The enzyme glutaminase (GLS1) is currently in clinical trials for oncology, yet there are no clear diagnostic criteria to identify responders. The evaluation of 25 basal breast lines expressing GLS1, predominantly through its splice isoform GAC, demonstrated that only GLS1-dependent basal B lines required it for maintaining de novo glutathione synthesis in addition to mitochondrial bioenergetics. Drug sensitivity profiling of 407 tumor lines with GLS1 and gamma-glutamylcysteine synthetase (GCS) inhibitors revealed a high degree of co-dependency on both enzymes across indications, suggesting that redox balance is a key function of GLS1 in tumors. To leverage these findings, we derived a pan-cancer metabolic signature predictive of GLS1/GCS co-dependency and validated it in vivo using four lung patient-derived xenograft models, revealing the additional requirement for expression of GAC above a threshold (log2RPKM + 1 ≥ 4.5, where RPKM is reads per kilobase per million mapped reads). Analysis of the pan-TCGA dataset with our signature identified multiple indications, including mesenchymal tumors, as putative responders to GLS1 inhibitors.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2017-156

Activating mutations in PIK3CA are commonly found in a wide variety of human cancers, and the dysregulation of the phosphoinositide-3 kinase (PI3K) signaling pathway has been implicated in tumor cell growth and survival. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent in cancer cells bearing PIK3CA mutants than those with wildtype PIK3CA. Preclinical studies demonstrate that taselisib induces more apoptotic cell death in PIK3CA mutant cancer cells than other PI3K inhibitors. We have discovered that taselisib has a dual mechanism of action, both blocking PI3K signaling, and inducing a decrease in p110a protein levels. Mass spec analysis reveals that taselisib treatment leads to the specific depletion of mutant p110alpha without significant change in wildtype p110alpha protein levels. This drug-induced p110a protein depletion is rescued by E1 inhibitors and by proteasome inhibitors. Other clinical PI3K inhibitors, including p110alpha selective and pan-PI3K inhibitors, are unable to induce the depletion of mutant p110 alpha protein. Furthermore, we have discovered that taselisib more effectively maintains pathway suppression in PIK3CA mutant cells at 24 hrs in response to feedback. In comparison to other clinical-stage PI3K inhibitors administered at a maximum tolerated dose, taselisib has superior efficacy with increased tumor regressions in PIK3CA mutant xenograft models. In summary, these preclinical studies indicate that PI3K inhibitors, which have the ability to trigger degradation of mutant p110a protein, can more effectively suppress the signaling pathway, which may result in greater anti-tumor activity and improved therapeutic index in PIK3CA mutant tumors. Citation Format: Kyung W. Song, Kyle A. Edgar, Donald S. Kirkpatrick, Lilian Phu, Stephen Schmidt, Michelle Nannini, Rebecca Hong, Eric Cheng, Lisa Crocker, Amy Young, Deepak Sampath, Lori Friedman. The PI3K inhibitor, taselisib, has a unique mechanism of action that leads to enhanced potency in PIK3CA mutant models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 146. doi:10.1158/1538-7445.AM2017-146

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. Taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha, sparing inhibition of p110beta, is more potent against cancer cells bearing mutations in the PIK3CA gene than those with wildtype PIK3CA. The mechanism leading to this enhanced mutant selectivity is revealed in these preclinical studies. Uniquely among PI3K inhibitors, taselisib has a gain of potency in PIK3CA mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PIK3CA mutant cells. In PIK3CA mutant cell culture-derived and patient-derived xenograft (PDX) models taselisib induces tumor regressions. In comparison to other clinical-stage PI3K inhibitors, taselisib confers superior anti-tumor activity in PIK3CA mutant xenografts when treated at a Maximum Tolerated Dose (MTD) in vivo. We have discovered that taselisib has a dual mechanism of action, both blocking kinase signaling and inducing down-regulation of the mutant p110alpha protein level in a dose-dependent and time-dependent manner. Taselisib treatment leads to the specific degradation of mutant p110alpha without significant change in wildtype p110alpha protein in cultured cells and in mutant xenograft models including PDX. Other clinical PI3K inhibitors, including PI3Kalpha selective and pan-PI3K inhibitors are unable to induce degradation of mutant p110 alpha. The taselisib-induced degradation of mutant p110a protein is ubiquitin-mediated and proteasome-dependent. These unique mechanistic effects of taselisib are most pronounced when comparing signaling suppression and p110a protein levels at 24 hours vs. 1 hour of drug exposure in PIK3CA mutant cell lines. This discovery indicates that PI3K inhibitors which trigger degradation of mutant p110a protein can more effectively suppress the signaling pathway in response to feedback, and may result in greater activity and improved therapeutic index. Citation Format: Friedman LS, Edgar KA, Song K, Schmidt S, Kirkpatrick DS, Phu L, Nannini MA, Hong R, Cheng E, Crocker L, Young A, Sampath D. The PI3K inhibitor, taselisib, has enhanced potency in PIK3CA mutant models through a unique mechanism of action [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S6-04.

Although mitogen-activated protein (MAP)–extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non–small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. Mol Cancer Ther; 12(6); 853–64. ©2013 AACR.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutational activation of the PIK3CA gene. The dysregulation of this pathway has been implicated in tumor cell growth and survival, thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of p110alpha sparing inhibition of p110beta, is investigated in these preclinical studies. Taselisib demonstrates greater potency in cancer cell lines harboring activating mutations in PIK3CA vs. wild-type lines, and induces regressions at tolerated doses in both PIK3CA mutant xenograft and patient-derived xenograft (PDX) models. When comparing taselisib to other clinical-stage PI3K inhibitors at Maximum Tolerated Dose (MTD) in vivo, taselisib confers greater activity in PIK3CA mutant models, which may indicate a larger therapeutic index. Unlike other PI3K inhibitors, taselisib has a gain of potency in PI3K alpha mutant SW48 isogenic cells compared to wildtype SW48 parental cells. Pathway inhibition and increased apoptosis are associated with the enhanced activity observed in PI3K alpha mutant cells. Other clinical PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not have improved potency in PI3K alpha mutant cells due to their inability to maintain pathway suppression after alleviation of negative feedback. The unique mechanism of action of taselisib is most notable when comparing signaling suppression at 24 hours vs. 1 hour of drug exposure. In mutant cells, Taselisib displays greater pathway suppression at 24 hours and is more effective at maintaining pathway suppression upon re-activation of growth factor RTK signaling. This mechanism is specific to PIK3CA mutant cells and not observed in wildtype cells. Ongoing studies to further elucidate this mechanism of action will be presented. Citation Format: Kyle A. Edgar, Kyung Song, Stephen Schmidt, Don Kirkpatrick, Lilian Phu, Michelle Nannini, Rebecca Hong, Eric Cheng, Amy Young, Deepak Sampath, Lori S. Friedman. The PI3K inhibitor, taselisib (GDC-0032), has enhanced potency in PIK3CA mutant models through a unique mechanism of action. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 370.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene or loss of the tumor suppressor PTEN. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis; thus PI3K is a promising therapeutic target with multiple inhibitors in clinical trials. The current study investigates preclinical mechanism of action of taselisib (GDC-0032), a novel, oral, selective inhibitor of PI3K alpha sparing inhibition of PI3K beta. In preclinical studies, taselisib demonstrates greater potency in cancer cell lines harboring activating PI3K alpha mutations vs wild-type lines. Taselisib induces tumor growth arrest and regressions at tolerated doses in xenograft and patient-derived xenograft (PDX) tumor models bearing PI3K alpha mutations. Notably, taselisib is distinguished from other PI3K inhibitors by enhanced potency in PI3K alpha mutant isogenic cells compared to parental cells. Pathway inhibition and apoptosis are associated with the enhanced activity of taselisib in PI3K alpha mutant cells. Other PI3K inhibitors, including PI3K alpha selective and pan-PI3K inhibitors, do not achieve the same level of activity in PI3K alpha mutant cell lines. Taselisib is more effective at maintaining suppression of the signaling pathway upon activation of Receptor Tyrosine Kinases (RTKs), such as after alleviation of negative feedback within the pathway. Additionally, a screen to evaluate secreted factors contributing to pan-PI3K inhibitor resistance was carried out in SW48 parental and PI3Kα H1047R isogenic cells. Growth factor ligands for the EGFR family were found to reduce the activity of PI3K inhibitors. Taken together, taselisib offers greater preclinical activity in PI3K alpha mutant cells when compared to other PI3K inhibitors, which may indicate a wider therapeutic index. Citation Format: Kyle A. Edgar, Michelle Nannini, Rebecca Hong, Charlie Eigenbrot, Stephen Schmidt, Amy Young, Deepak Sampath, Jeffrey J. Wallin, Lori S. Friedman. Characterization of the enhanced potency of PI3K inhibitor taselisib (GDC-0032) in PI3K mutant cell lines and models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2672. doi:10.1158/1538-7445.AM2015-2672

Tumors exhibit altered metabolic profiles compared to normal, non-transformed tissues. In the presence of oxygen, most differentiated cells convert glucose to ATP by oxidative phosphorylation (OX-PHOS), whereas tumor cells consume glucose and convert it to lactate, a phenomenon known as “aerobic glycolysis” or the “Warburg effect”. There is increased interest in targeting glycolysis as a therapeutic strategy in oncology, and one of the key enzymes in this pathway is lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate in the last step of glycolysis. A small molecule LDHA inhibitor that was identified by high throughput screening was optimized to generate GNE-140, which has a biochemical EC50 of 0.005 μM and inhibits MiaPaCa-2 pancreatic cell proliferation with an EC50 of 0.25 μM. We show that inhibition of LDHA reduces carbon flux from pyruvate to lactate, impairs glycolysis, and leads to global effects in cell metabolism. Cells cease proliferation within 24 hours of LDHA inhibition, yet only undergo cell death following sustained pathway inhibition. Finally, we show that pancreatic cell lines that are resistant to LDHA inhibition can be re-sensitized to LDHA inhibition by inhibiting OX-PHOS. In conclusion, we describe a potent cell active LDHA inhibitor that modulates glycolysis, induces cell death, and that can be used to help evaluate the importance of LDHA in tumor growth. Citation Format: Thomas O'Brien, Hans Purkey, Anna Hitz, Dave Peterson, Aaron Boudreau, Christopher Delnagro, Mandy Kwong, Rebecca Hong, Min Gao, Jodi Pang, Alex Vanderbilt, Simon Williams, Laurent Salphati, Deepak Sampath, Georgia Hatzivassiliou, Marie Evangelista. Inhibiting glycolysis with an LDHA inhibitor: A new solution to an old problem. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2014-964

Targeting cancer metabolism is currently being evaluated as a potential therapeutic target. Identification of tumor subtypes with specific metabolic requirements will enable the success of such therapies. Using cell line models of pancreatic ductal adenocarcinoma (PDAC), we conducted broad metabolite profiling and identified 3 metabolic subtypes through non-negative matrix factorization (NMF) consensus clustering. Among the most distinguishing features between the subtypes were levels of metabolites important for lipid synthesis, amino acid, and energy-related pathways, suggestive of distinct metabolic dependencies. Using RNAseq we show that these subtypes are defined at the transcriptional level and are associated with known subtypes of PDAC. In metabolic flux experiments, we also show that glucose and glutamine are differentially utilized by each subtype, and as a consequence, exhibit distinct sensitivity to inhibition of various metabolic pathways. Finally, through screening of a large panel of tumor cell lines with various metabolic inhibitors, we cross-validate our finding in PDAC and provide strong evidence that these same subtypes exist and can be identified in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity to metabolic inhibitors in cancer cells. Citation Format: Anneleen Daemen, David Peterson, Nisebita Sahu, Ron McCord, Kaska Kowanetz, Anna Hitz, Xiangnan Du, Bonnie Liu, Min Gao, John Moffat, Rebecca Hong, Deepak Sampath, Mark Merchant, Thomas O'Brien, Bob Yauch, Jeff Settleman, Jing Qing, Georgia Hatzivassiliou, Marie A. Evangelista. Identification of distinct metabolic subtypes within pancreatic ductal adenocarcinoma through broad metabolite profiling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2014-1430

Background: Akt, a serine/threonine protein kinase, is a key signaling node in the PI3K/Akt/mTOR pathway and plays an essential role in regulating tumor cell proliferation, migration and survival. PI3K/Akt signaling is up-regulated in gastric cancer, mainly due to loss of PTEN, and is associated with poor prognosis and chemoresistance. Chemotherapeutics such as doxorubicin and 5-FU/cisplatin have been shown to upregulate PI3K/Akt signaling in several solid cancers in response to DNA damage. We have previously reported on a potent highly selective ATP-competitive pan-Akt inhibitor, GDC-0068, which inhibits cell cycle progression and viability of cancer cell lines and xenografts driven by Akt signaling, including those with defects in the tumor suppressor PTEN, oncogenic mutations in PIK3CA, and amplification of HER2. Thus, we hypothesized that the combination of FOLFOX and GDC-0068 would induce anti-tumor activity in primary gastric cancer models. Methods: Gastric cancer cell lines (n=11) were treated with increasing concentrations of GDC-0068 and FOLFOX and evaluated for combinational efficacy over single agent via cell viability assay. For in vivo studies, primary gastric cancer xenograft models were implanted in immuno-compromised mice and treated with GDC-0068 and FOLFOX as single agents or in combination. Results: In vitro, the combination of GDC-0068 and FOLFOX results in enhanced inhibition of tumor cell viability in the cell lines with activation of the PI3K pathway compared to either single agent alone. These results were recapitulated in vivo, in the primary xenograft models STO#240 and STO#182, where the combination of GDC-0068 and FOLFOX resulted in increased tumor growth inhibition or regressions compared to single agents alone. All combinations tested were well tolerated in vivo based on minimal changes in body weights. Conclusions: Our preclinical studies demonstrate improved anti-tumor efficacy when GDC-0068 is combined with FOLFOX in gastric cancer models in vitro and in vivo. The data supports the clinical development of GDC-0068 in combination with FOLFOX for the treatment of gastric cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B190. Citation Format: Ellen Ingalla, Rebecca Hong, Heidi Savage, Elizabeth Punnoose, Sandra Rost, Hartmut Koeppen, Deepak Sampath, Michelle A. Nannini. GDC-0068 is a novel and selective Akt inhibitor that enhances the efficacy of FOLFOX in primary gastric cancer models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B190.

Endocrine therapies such as letrozole or fulvestrant are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but patients ultimately relapse. Phosphatidylinositol 3-kinases (PI3K) regulate breast tumor cell growth, migration and survival. The alpha isoform of PI3K is frequently mutated and activated in HR+ breast cancer and has been implicated in resistance to endocrine therapies. PI3K inhibitors are therefore attractive for combination with endocrine therapies. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Preclinical data show that GDC-0032 has increased activity against PI3K alpha isoform (PIK3CA) mutant and HER2-amplified cancer cell lines. Single agent and combination studies were carried out to determine if GDC-0032 enhances the anti-tumor activity of endocrine therapies in human breast cancer models. MCF7 cells (Estrogen Receptor positive (ER+), PI3K alpha E545K mutant) were transfected with the aromatase gene and stable clones (MCF7-ARO) were selected that are capable of converting androstenedione to estrogen in culture. When grown in the presence of androstenedione, MCF7-ARO cells were more reliant on estrogen for growth. Under these conditions the cells were treated with GDC-0032 in combination with endocrine therapies and assayed for cellular viability, modulation of PI3K pathway and ER pathway markers and apoptosis induction. The combination of GDC-0032 and endocrine therapies decreased the cellular viability of MCF7-ARO cells and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggests a mechanism of action for the combination. The data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for HR+ breast cancer treatment in the clinic. Citation Format: Jeffrey J. Wallin, Jane Guan, Rebecca Hong, Carol O'Brien, Timothy Wilson, Michelle Nannini, Deepak Sampath, Marcia Belvin, Lori S. Friedman. GDC-0032 PI3K inhibitor enhances the efficacy of endocrine therapies in breast cancer cells by differentially regulating the expression of ER-targeted genes and increasing apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 546. doi:10.1158/1538-7445.AM2013-546

Glutaminase is a key enzyme for the conversion of glutamine to glutamate with subsequent use as a fuel in the TCA cycle, glutathione production and protein synthesis. A subset of tumor cells shows preferential dependency on glutamine and glutaminase for cell proliferation and survival, consistent with “metabolic addiction” to this pathway. Between two glutaminase loci in the genome, the gene for kidney-type glutaminase (Gls1) has 2 alternatively spliced products (KGA and GAC), with distinct regulation and expression from liver-type glutaminase (Gls2). Furthermore, based on distinct C-termini and enzymatic properties, it has been proposed that the Gls1 alternatively spliced isoforms (KGA and GAC) may themselves have distinct functions. Our data further elucidates the role of glutaminase in cancer by addressing the following key questions: a) what is the major role of glutaminase in cells most sensitive to its inhibition? b) is GAC interchangeable with KGA in its expression pattern and role in tumor cells? c) how do tumors adapt to glutaminase inhibition? By generating and using a panel of GAC and KGA isoform-selective reagents we show that the two isoforms have distinct patterns of expression and distinct roles in cell proliferation, migration and tumor growth in vivo. In addition, based on metabolomics, functional and complementation assays, we propose a mechanistic basis for tumor cell dependency on glutaminase and the tumor adaptive response to glutaminase inhibition. Our data reveal the basis for the addiction of tumor subsets to glutaminase and elucidate the role of distinct isoforms of kidney-type glutaminase isoforms in cancer. Citation Format: Bonnie Liu, Kyung Song, Mandy Kwong, Min Gao, Rebecca Hong, Michelle Nannini, Deepak Sampath, Marcia Belvin, David Peterson, Marie Evangelista, Anneleen Daemen, Ron Firestein, Georgia Hatzivassiliou. Elucidating the role of distinct glutaminase isoforms in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2013-4618