Phosphatidylinositol 3-Kinase -Selective Inhibition With Alpelisib (BYL719) in PIK3CA-Altered Solid Tumors: Results From the First-in-Human Study

PurposeWe report the first-in-human phase Ia study to our knowledge (ClinicalTrials.gov identifier: NCT01219699) identifying the maximum tolerated dose and assessing safety and preliminary efficacy of single-agent alpelisib (BYL719), an oral phosphatidylinositol 3-kinase (PI3K)-selective inhibitor.Patients and MethodsIn the dose-escalation phase, patients with PIK3CA-altered advanced solid tumors received once-daily or twice-daily oral alpelisib on a continuous schedule. In the dose-expansion phase, patients with PIK3CA-altered solid tumors and PIK3CA-wild-type, estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer received alpelisib 400 mg once daily.ResultsOne hundred thirty-four patients received treatment. Alpelisib maximum tolerated doses were established as 400 mg once daily and 150 mg twice daily. Nine patients (13.2%) in the dose-escalation phase had dose-limiting toxicities of hyperglycemia (n = 6), nausea (n = 2), and both hyperglycemia and hypophosphatemia (n = 1). Frequent all-grade, treatment-related adverse events included hyperglycemia (51.5%), nausea (50.0%), decreased appetite (41.8%), diarrhea (40.3%), and vomiting (31.3%). Alpelisib was rapidly absorbed; half-life was 7.6 hours at 400 mg once daily with minimal accumulation. Objective tumor responses were observed at doses 270 mg once daily; overall response rate was 6.0% (n = 8; one patient with endometrial cancer had a complete response, and seven patients with cervical, breast, endometrial, colon, and rectal cancers had partial responses). Stable disease was achieved in 70 (52.2%) patients and was maintained > 24 weeks in 13 (9.7%) patients; disease control rate (complete and partial responses and stable disease) was 58.2%. In patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer, median progression-free survival was 5.5 months. Frequently mutated genes ( 10% tumors) included TP53 (51.3%), APC (23.7%), KRAS (22.4%), ARID1A (13.2%), and FBXW7 (10.5%).ConclusionAlpelisib demonstrated a tolerable safety profile and encouraging preliminary activity in patients with PIK3CA-altered solid tumors, supporting the rationale for selective PI3K inhibition in combination with other agents for the treatment of PIK3CA-mutant tumors

Loss of serum and glucocorticoid-regulated kinase 3 (SGK3) does not affect proliferation and survival of multiple myeloma cell lines.

Multiple myeloma (MM) is a generally fatal plasma cell cancer that often shows activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. Targeted pharmacologic therapies, however, have not yet progressed beyond the clinical trial stage, and given the complexity of the PI3K/Akt signalling system (e.g. multiple protein isoforms, diverse feedback regulation mechanisms, strong variability between patients) it is mandatory to characterise its ramifications in order to better guide informed decisions about the best therapeutic approaches. Here we explore whether serum and glucocorticoid-regulated kinase 3 (SGK3), a potential downstream effector of PI3K, plays a role in oncogenic signalling in MM cells--either in concert with or independent of Akt. SGK3 was expressed in all MM cell lines and in all primary MM samples tested. Four MM cell lines representing a broad range of intrinsic Akt activation (very strong: MM.1s, moderate: L 363 and JJN-3, absent: AMO-1) were chosen to test the effects of transient SGK3 knockdown alone and in combination with pharmacological inhibition of Akt, PI3K-p110α, or in the context of serum starvation. Although the electroporation protocol led to strong SGK3 depletion for at least 5 days its absence had no substantial effect on the activation status of potential downstream substrates, or on the survival, viability or proliferation of MM cells in all experimental contexts tested. We conclude that it is unlikely that SGK3 plays a significant role for oncogenic signalling in multiple myeloma

SGK3 knockdown in the broader context of MEK1,2/PI3K blockade.

<p>MM cells were electroporated with stealth siRNAs against EGFP or SGK3 and drugs were added at day 2 post-electroporation for a further 3-day incubation. Cell death was measured by annexin V/PI staining and FACS analysis. Error bars denote s.e.m. based on 3 independent experiments. The survival rates were calculated relative to DMSO-treated cells. The absolute survival rates for the experimental pairs in these experiments (DMSO treated stEGFP vs. DMSO treated stSGK3 transfected cells) were 92.7% vs. 93.6% (AMO-1) and 92.7% vs. 91.9% (L-363), i.e. there was no substantial difference between control cells and SGK3 knockdown cells. Titration of BYL-719 and choice of its concentration are detailed in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122689#pone.0122689.ref015" target="_blank">15</a>]. Of note, the strong synergistic effect observed in AMO-1 cells for the combination of PI3K-p110α inhibitor BYL-719 and MEK1,2 inhibitor PD0325901 is not observed when the PI3K-p110α inhibitor is substituted with an Akt inhibitor (MK-2206 or Akti1,2), but it is also not mirrored by the combination of MEK1,2 inhibition and SGK3 depletion.</p

SGK3 knockdown in MM cell lines.

<p>A) Western blot showing time-dependence of stealth siRNA-mediated SGK3 (stSGK3) depletion in electroporated JJN-3 cells. A stealth siRNA against enhanced green fluorescent protein (stEGFP) was used in control electroporations. B) SGK3 knockdown and phosphorylation levels of Akt and of potential SGK3 downstream substrates in stEGFP vs. stSGK3 transfected MM cell lines. Cells were harvested at day 3 post-electroporation. For each cell line two gels were run and the representative β-actin control derives from the gel that was also stained for P-Akt (Thr308) (all cell lines) and pan-Akt (L-363, MM.1s) or P-FOXO1/3A and P-GSK-3β (JJN-3). C) SGK3 knockdown does not substantially affect the survival of MM cells. Shown are the results of annexin V/PI staining followed by FACS analysis at day 4 post-electroporation. Relative survival rates are shown, i.e. survival (the annexin V/PI negative fraction) in stEGFP transfected control samples was always set as 100% and survival of the respective SGK3 knockdown samples is shown relative to their cognate controls. At least three independent experiments were performed for each cell line. Error bars denote s.e.m. D) Same experiments as described for C) but viability determined by alamarBlue colorimetric assay. E) Proliferation analysis (one of 2 similar experiments shown) performed on day 4 post-electroporation in MM cell lines treated with the stealth siRNA against EGFP versus stealth siRNA against SGK3. The percentages given above the lines denote the respective share of cells that have incorporated bromo-desoxyuridine (BrdU) during a 2 h BrdU pulse, indicating active DNA synthesis. F) Forward-scatter analysis of stEGFP versus stSGK3 treated cells at day 4 post-electroporation as a measure of cell volume. Top: examples for selection of the live cell fractions of stEGFP and stSGK3 transfected cells based on their forward versus sideward scatter location. Bottom: Distribution of the mean forward scatter values for between 4 and 5 independent experiments (i.e. different electroporations) per cell line. Horizontal black lines mark the mean value of the datapoints shown and vertical black lines indicate the s.e.m.</p

Serum dependence of SGK3 depleted MM cells.

<p>MM cells transfected with either stealth siRNA against EGFP (white columns) or against SGK3 (grey columns) were washed three times with PBS at day 2 post-electroporation and resuspended in fresh medium, subsequently adjusted to contain the indicated concentrations of FBS. After further culture for 3 days cell death was determined by annexin V/PI staining and FACS analysis. Error bars indicate s.e.m. based on 4 independent experiments.</p

SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in MM cell lines.

<p>Shown are Western blots for PI3K pathway-associated signalling proteins or for their phosphorylated forms. Cells from the MM cell lines indicated were harvested from standard cell culture, the signals are thus representative of steady-state levels in culture. One cell lysate per line was used to load multiple gels. The representative β-actin control derives from the same blot on which SGK3 and P-FOXO1/3A were also stained. Note: the strong phospho-STAT3 signal in INA-6 cells results from permanent supplementation of the culture with recombinant human IL-6.</p

SGK3 expression in relation to (activated) signalling components of the PI3K/Akt system in primary MM cells.

<p>Shown are Western blots prepared from frozen pellets of primary MM cells purified by CD138 microbead selection. The material was used to load two gels, with the representative β-actin control belonging to the same blot on which PI3K-p110α, P-FOXO1/3A, P-Akt (Thr308) and pan-Akt were also stained. Of note, the phospho-Akt (Thr308) antibody also stained a slightly larger band in most primary MM samples (marked by "?") that was not visible in any MM cell line. This band runs slightly higher, though, than the SGK3 band as stained on the parallel blot. Staining for P-PRAS40 (CST; no. 2997) was performed after staining for P-GSK-3β. Since both antibodies were raised in rabbit the latter signal reappeared in the P-PRAS40 blot.</p

Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function

Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCR, CD19, CD14, SYTOX ® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions

SGK3 knockdown in combination with Akt inhibition.

<p>A) Titration of allosteric Akt inhibitors MK-2206 and Akti1,2. MM cells were incubated with the drugs for 30 min prior to harvest for Western blotting. B) Dose-effect curves representing cell death (annexin V) or viability (alamarBlue) assays for MM.1s cells (left), L-363 cells (middle) and AMO-1 cells (right) from either untreated cultures (blue dots), cells electroporated with stealth siRNA against EGFP (green dots) or cells electroporated with stealth siRNA against SGK3 (red dots), and treated with various concentrations of MK-2206 for 3 days. Drugs were added to electroporated cells at day 2 post-electroporation. Cells from untreated cultures were kept at similar densities as those of electroporated cells prior to drug addition. Each dose/effect curve is based on between 3 and 4 independent experiments. Error bars indicate s.e.m. C) Same experimental setup as described in B) but with Akt inhibitor Akti1,2. Each dose/effect curve is based on between 2 and 3 independent experiments. Error bars indicate s.e.m.</p