Molecular quantification of tissue disease burden is a new biomarker and independent predictor of survival in mastocytosis

A high allele burden of the KIT D816V mutation in peripheral blood or bone marrow aspirates indicates multi-lineage hematopoietic involvement and has been associated with an aggressive clinical course of systemic mastocytosis. Since mast cells are substantially underrepresented in these liquid specimens, their mutation burden likely underestimates the tumor burden of the disease. We used a novel previously validated digital polymerase chain reaction (PCR) method for KIT D816V analysis to systematically analyze the mutation burden in formalin-fixed, paraffin-embedded bone marrow tissue sections of 116 mastocytosis patients (91 with indolent and 25 with advanced systemic mastocytosis), and to evaluate for the first time the clinical value of the tissue mutation burden as a novel biomarker. The KIT D816V mutation burden in the tissue was significantly higher and correlated better with bone marrow mast cell infiltration (r=0.68 vs. 0.48) and serum tryptase levels (r=0.68 vs. 0.58) compared to that in liquid specimens. Furthermore, the KIT D816V tissue mutation burden was: (i) significantly higher in advanced than in indolent systemic mastocytosis (P=0.001); (ii) predicted survival of patients in multivariate analyses independently; and (iii) was significantly reduced after response to cytoreductive therapy. Finally, digital PCR was more sensitive in detecting KIT D816V in bone marrow sections of indolent systemic mastocytosis patients than melting curve analysis after peptide nucleic acid-mediated PCR clamping (97% vs. 89%;

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

CDK4/CDK6 Inhibitors Synergize with Midostaurin, Avapritinib, and Nintedanib in Inducing Growth Inhibition in <i>KIT</i> D816V⁺ Neoplastic Mast Cells

In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials

STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

International audienc

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis

<div><p>A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after <i>toll-like</i> receptor (TLR)-activation and glucose-deprivation or co-treatment with 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an <i>in-vivo</i> mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our <i>in vitro</i> observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone <i>Listeria monocytogenes</i> mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.</p></div

Impact of AMPK activators on IL-12p40/IL-10 induction.

<p>Human monocytes were preincubated for 90 minutes with different concentrations of A-769662 (a, b) or AICAR (c, d) or medium and then stimulated with LPS. Secretion of IL-10 (a, c) and IL-12p40 (b, d) was determined from 20 hr culture supernatants by ELISA. Data are representative of 3–5 independent experiments and presented as % response ± SD. A-769662 treatment alone induced no significant cytokine production: IL-10: 4 times below detection level, IL-12p40: 3 times below detection level 1x 1.8 pg/mL, Similarly, AICAR treatment alone induced no significant cytokine production: IL-10: 3x ≤ 3.1 pg/mL, IL-12p40: 3 times below detection level. *p ≤ 0.05, ***p ≤ 0.01.</p

Modulation of cytokine production by 2-DG in murine BMDMs.

<p>BMDMs from BALB/c mice were cultured in 6 well plates and treated with 1, 3, and 5 mM 2-DG and <i>L</i>.<i>m</i>. as indicated. IL-10 (a) and IL-12p40 (b) were determined from 20 hour culture supernatants by ELISA. *p ≤ 0.05, **p ≤ 0.01 <i>L</i>.<i>m</i>. <i>Listeria monocytogenes</i>. 2-DG treatment alone induced the following cytokine levels: IL-10: 4 times ≤ 84.5 pg/mL, IL12p40: 4 times ≤ 5.1 pg/mL.</p

2-DG protects mice from Listeria monocytogenes <i>(L</i>.<i>m</i>.<i>)</i> infection.

<p>BALB/c mice (n = 5) were pretreated i.p. with 2-DG or PBS for 3 days and then challenged with 5 x 10⁴ <i>L</i>.<i>m</i>. On day 3 after infection the number of bacteria in the spleen and the liver were determined (a). Liver histomorphology was analyzed by H&E staining on day 3 after infection (b). Granulomatous lesions are indicated (by arrows). **p ≤ 0.005. (c) Livers (n = 3 per group) were analyzed by immunohistochemical staining of F4/80 on day 3 post infection. Representative images from each group of mice are shown (brown color, F4/80 staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: 50 μm. Inserts: the high-power views. (d) Quantitative analysis of F4/80 staining in the liver specimens using HistoQuest software. The box-plot analyses represent the number of F4/80-positive cells (designated as “Count”) and also the total and mean areas of F4/80-positive cells (designated as “Total Area” and “Mean Area”, respectively); the total area values reflect both the magnitude and morphology/size of positive cells, while the mean area values reflect predominantly the morphology/size of positive cells. ***p ≤ 0.001; only significant p values are shown.</p

2-DG differentially modulates pro- and anti-inflammatory cytokine secretion in human monocytes.

<p>Human monocytes were preincubated for 90 minutes with different concentrations of 2-DG or medium and then stimulated with LPS. Secretion of IL-10 (a) and IL-12p40 (b) IL-23p19 (c), IL-1β (d), IL-6 (e) TNF-α (f) was determined from 18–24 hr culture supernatants by ELISA. Pretreatment of monocytes with rapamycin (100 nM) served as control. Data are representative of 5 independent experiments and presented as % response ± SD. In unstimulated cultures cytokines were undetectable. 2-DG treatment alone induced no significant cytokine production: IL-10: 2 times below detection level, 3 times ≤ 1.4 pg/mL, IL-12p40: 4 times below detection level, 1x 3.8 pg/mL, IL-23p19: 5 times below detection level, IL-1β: 2 times below detection level, 3 times ≤ 22.2 pg/mL, IL-6: 5 times below 11.6 pg/mL, TNF-α 1x below detection level 4 times ≤ 2.8 pg/mL; Mean cytokine levels after LPS stimulation in the absence of 2-DG were: IL-10, 904 ± 1096 pg/mL; IL-12p40, 535 ± 509 pg/mL; IL-23p19, 136 ± 152 pg/mL; IL-1β, 9471 ± 10692 pg/mL; IL-6, 1608 ± 414 pg/mL; TNF-α, 1596 ± 642 pg/mL. *p ≤ 0.05, ***p ≤ 0.005.</p

Modulation of inflammatory mediators by 2-DG at the transcriptional level and its impact of 2-DG on mTOR signaling in human monocytes.

<p>mRNA levels of IL-10, IL-12B(p40), IL-23A(p19) in human monocytes were assessed by real-time PCR analysis. Monocytes were pre-treated with 2-DG for 90 minutes or medium and then stimulated with LPS for 4 hr. Expression levels were normalized to house keeping genes and shown relative to LPS-stimulated cells. Data are displayed as means ± SD of two independent experiments. Significance was assessed by two-tailed t test; ** p ≤ 0.01 (a). Monocytes were preincubated for 15 minutes with respective mM doses of 2-DG, rapamycin (100 nM) or medium as indicated and then stimulated with LPS. Whole cell lysates were analyzed by immunoblotting using specific antibodies against phospho-S6RP (b)<b>.</b> Data are representative of 2 independent experiments.</p