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

Keratinocyte growth factor impairs human thymic recovery from lymphopenia

Background: The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment of relapsing-remitting multiple sclerosis (RRMS); however 50% of patients develop novel autoimmunity post-treatment. Most at risk are individuals who reconstitute their T-cell pool by proliferating residual cells, rather than producing new T-cells in the thymus; raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in non-human primates. Methods: Following a dose-tolerability sub-study, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5·0; with ≥2 relapses in the previous 2 years) were randomised to placebo or 180mcg/kg/day palifermin, given for 3 days immediately prior to and after each cycle of alemtuzumab, with repeat doses at M1 and M3. The interim primary endpoint was naïve CD4+ T-cell count at M6. Exploratory endpoints included: number of recent thymic-emigrants (RTEs) and signaljoint T-cell receptor excision circles (sjTRECs)/mL of blood. The trial primary endpoint was incidence of autoimmunity at M30. Findings: At M6, individuals receiving palifermin had fewer naïve CD4+T-cells (2.229x107 /L vs. 7.733x107 /L; p=0.007), RTEs (16% vs. 34%) and sjTRECs/mL (1100 vs. 3396), leading to protocoldefined termination of recruitment. No difference was observed in the rate of autoimmunity between the two groups Conclusion: In contrast to animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/haematology setting where alemtuzumab is often used as part of the conditioning regime.Trial - MRC and Moulton Trust Funding Me (senior Author) - Wellcome Trust Funding

Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy

In a proportion of patients with chronic myeloid leukemia (CML) being treated with dasatinib, we recently observed large granular lymphocyte (LGL) expansions carrying clonal T-cell receptor (TCR) gamma/delta gene rearrangements. To assess the prevalence and role of clonal lymphocytes in CML, we collected samples from patients (n = 34) at the time of diagnosis and during imatinib and dasatinib therapies and analyzed lymphocyte clonality with a sensitive polymerase chain reaction-based method of TCR gamma and delta genes. Surprisingly, at CML diagnosis, 15 of 18 patients (83%) had a sizeable clonal, BCR-ABL1 negative lymphocyte population, which was uncommon in healthy persons (1 of 12; 8%). The same clone persisted at low levels in most imatinib-treated patients. In contrast, in a distinct population of dasatinib-treated patients, the diagnostic phase clone markedly expanded, resulting in absolute lymphocytosis in blood. Most patients with LGL expansions (90%) had TCR delta rearrangements, which were uncommon in patients without an LGL expansion (10%). The TCR delta clones were confined to gamma delta(+) T- or natural killer-cell compartments and the TCR gamma clones to CD4(+)/CD8(+) alpha beta(+) fractions. The functional importance of clonal lymphocytes as a part of leukemia immune surveillance and the putative anergy- reversing role of dasatinib require further evaluation. (Blood. 2010; 116(5): 772- 782

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 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

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