Erythropoietin Down-Regulates Stem Cell Factor Receptor (Kit) Expression in the Leukemic Proerythroblast: Role of Lyn Kinase

Overexpression of the transcription factor Spi-1/PU.1 by transgenesis in mice induces a maturation arrest at the proerythroblastic stage of differentiation. We have previously isolated a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in the presence of either erythropoietin (Epo) or stem cell factor (SCF). Using these cell lines, we observed that EpoR stimulation by Epo down-regulated expression of the SCF receptor Kit and induced expression of the Src kinase Lyn. Furthermore, enforced expression of Lyn in the cell lines increased cell proliferation in response to Epo, but reduced cell growth in response to SCF in accordance with Lyn ability to down-regulate Kit expression. Together, the data suggest that Epo-R/Lyn signaling pathway is essential for extinction of SCF signaling leading the proerythroblast to strict Epo dependency. These results highlight a new role for Lyn as an effector of EpoR in controlling Kit expression. They suggest that Lyn may play a central role in during erythroid differentiation at the switch between proliferation and maturation

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies.

Duvelisib is an orally active dual inhibitor of PI3K-δ and PI3K-γ in clinical development in hematologic malignancies (HM). To identify novel pairings for duvelisib in HM, it was evaluated alone and in combination with 35 compounds comprising a diverse panel of standard-of-care agents and emerging drugs in development for HM. These compounds were tested in 20 cell lines including diffuse large B-cell, follicular, T-cell, and mantle cell lymphomas, and multiple myeloma. Single agent activity was seen in fourteen cell lines, with a median GI50 of 0.59 μM. A scalar measure of the strength of synergistic drug interactions revealed a synergy hit rate of 19.3% across the matrix of drug combinations and cell lines. Synergy with duvelisib was prominent in lymphoma lines with approved and emerging drugs used to treat HM, including dexamethasone, ibrutinib, and the BCL-2 inhibitor venetoclax. Western blotting revealed that certain duvelisib-treated cell lines showed inhibition of phosphorylated (p) AKT at serine 473 only out to 12 hours, with mTORC2 dependent re-phosphorylation of pAKT evident at 24 hours. Combination with dexamethasone or ibrutinib, however, prevented this reactivation leading to durable inhibition of pAKT. The combination treatments also inhibited downstream signaling effectors pPRAS40 and pS6. The combination of duvelisib with dexamethasone also significantly reduced p-4EBP1, which controls cap dependent translation initiation, leading to decreased levels of c-MYC 6 hours after treatment. In support of the in vitro studies, in vivo xenograft studies revealed that duvelisib in combination with the mTOR inhibitor everolimus led to greater tumor growth inhibition compared to single agent administration. These data provide a rationale for exploring multiple combinations in the clinic and suggest that suppression of mTOR-driven survival signaling may be one important mechanism for combination synergy

Down-regulation of Kit expression by Epo.

<p>A: Kit and EpoR expression were studied in the 633, 663 and 812 cells grown continuously in the presence of either Epo (1 U/mL) or SCF (100 ng/mL) or a combination of Epo (1 U/mL)+SCF (100 ng/mL). Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. Western blots are from a representative experiment. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to β actin expression. The fold change in Kit expression between SCF-cultured cells and either Epo or Epo+SCF-cultured cells is indicated under Kit immunoblotting. B: Representative diagram of flow cytometry analysis showing Kit membrane expression in cells cultured with SCF (100 ng/mL; black line) or Epo+SCF (dotted line). Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of four independent experiments. C: AG490 inhibits the down-regulation of Kit by Epo. Cells were cultured for 48 hrs in a medium containing 10% FBS, 1 U/mL Epo and in the presence or absence (-) of AG490 (10 µM). Representative Western blot analysis of whole cell lysates with antibodies directed against Kit, Stat5 and phosphorylated Stat5. β-actin was used as loading control. The fold change in Kit expression between AG490-treated and untreated cells is indicated under Kit immunoblotting. Representative diagram of flow cytometry analysis showing Kit membrane expression in 663 and 812 cells cultured with Epo and treated (black line) or not (dotted line) with AG490 (10 µM) for 48 hrs. Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of positive cells in three independent experiments. D: Cells were cultured for 24, 48 and 72 hrs in the presence of a combination of Epo (1 U/mL)+SCF (100 ng/mL) or Epo (0.1 U/mL)+SCF (100 ng/mL) and viable cells were numbered. Data are mean±SD of five experiments, each performed in duplicate. E: The regulation of Kit expression is transcriptional: RT-PCR analysis of <i>Kit</i> and <i>EpoR</i> transcripts in 633, 663 and 812 cells cultured in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). cDNAs were amplified with specific primers for <i>Kit</i>, <i>EpoR</i> or <i>gapdh</i> as control.</p

Reversible down-regulation of Kit in response to Epo.

<p>Switch from Epo to SCF (A) and switch from SCF to Epo (B): 663 and 812 cells continuously cultured with Epo (1 U/mL) or SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with SCF (100 ng/mL) or Epo (1 U/mL), respectively. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. The fold change in Kit expression following the switch in cytokines is indicated under Kit immunoblotting. Representative diagrams of flow cytometry analysis showing Kit expression on the surface of cells expanded with Epo (dotted line) or SCF (black line). Control IgG profile is shown in grey. The tables indicate the mean fluorescence intensity (MFI)±SD of positive cells in at least four independent experiments. C: Switch from SCF to various concentrations of Epo. 663 cells cultured with SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with Epo at doses indicated. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Kit, EpoR and β-actin as a loading control. The fold decrease in Kit expression following the switch from SCF to various doses of Epo is indicated under Kit immunoblotting. Western blots are from a representative experiment repeated 3 times.</p

Epo controls Lyn expression.

<p>A: Lyn and EpoR expression were studied in 633, 663 and 812 cells continuously grown in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). Whole cell lysates were subjected to Western blot analysis with antibodies directed against Lyn, EpoR and β-actin as a loading control. B: Switch from SCF to Epo induces the expression of Lyn. 663 and 812 cells cultured with SCF (100 ng/mL) were extensively washed with medium without cytokine and then expanded for 48 hrs with Epo at the indicated doses. Whole cell lysates were subjected to Western blot analysis with antibodies directed against Lyn, Stat5, phosphorylated-Stat5 and β-actin as loading control. C: AG490 inhibits the expression of Lyn. 663 and 812 cells were cultured for 48 hrs in a medium containing 10% serum in the presence or absence of AG490 (10 µM) and in the presence of Epo (1 U/ml). Whole cell lysates were subjected to Western blot analysis with antibodies to Lyn, EpoR and β-actin. Western blots are from a representative experiment. D: RT-PCR analysis of <i>Lyn</i> transcription in 633, 663 and 812 cells cultured in the presence of either Epo (1 U/mL) or SCF (100 ng/mL). DNAs were amplified with specific primers for <i>Lyn</i> or <i>gapdh</i> as control.</p

Expression of Lyn^WT and Lyn^Y397F in 663 cells.

<p>(A) A clone of 663 cells stably transfected with pEF-neo Lyn^WT or pEF-Neo empty vector was expanded in the presence of Epo (1 U/mL) or SCF (100 ng/mL). Whole cell extracts were subjected to Western blot using anti-MT, anti-Lyn, anti-EpoR, anti-Kit antibodies and anti-β actin as a loading control. The fold increase in Lyn expression and the fold decrease in Kit expression between pEF-Neo and pEF-neo Lyn^WT transfected cells are indicated under Lyn or Kit immunoblotting. (B) 663 cells were transfected with pEF-neo Lyn^Y397F or pEF-Neo empty vector. Proliferation of 663 cells expressing Lyn^Y397F and cultured in the presence of Epo (1 U/mL). Whole cell extracts were subjected to Western blot using an anti-MT, anti-Lyn, an EpoR, an anti-Kit and an anti-β actin antibody as a loading control. The fold increases in Lyn and Kit expressions between pEF-Neo and pEF-neo Lyn^Y397F transfected cells are indicated under Lyn or Kit immunoblotting. C: Transfected 663-neo and 663-Lyn^WT cells were plated at 2×10⁵ cells/mL in medium containing Epo or SCF at concentrations indicated or with no cytokine. Viable cells were scored after 48 hours. Data are mean±SD of five independent experiments performed in triplicate. *indicates statistical significance by student <i>t</i> test: P<.05 compared with the control neo-cells. D: Transfected 663-neo and 663-Lyn^Y397F cells were plated at 2×10⁵ cells/mL in the presence of increasing concentrations of Epo and viable cells were scored after 48 hours. Data are mean±SD of five independent experiments performed in triplicate. Results are shown for one 663-Lyn^WT clone and one 663-Lyn^Y397F clone. Experiments were performed with another clone in each category and similar results were obtained. *indicates statistical significance by student <i>t</i> test: P<.05 compared with the control neo-cells.</p

Proliferation of <i>spi-1</i> transgenic proerythroblasts and expression of EpoR, Kit and Stat5.

<p>A: Cells were continuously cultured in the presence of Epo (1 U/mL) or SCF (100 ng/mL). Number of living cells was monitored at 24, 48 and 72 hours using the Trypan blue exclusion staining and a Vi-Cell analyzer (Becton Coulter). Mean number of living cells and standard deviations were determined from 3 independent experiments performed in duplicate. B: Representative Western blot of lysates from cells grown with Epo (1 U/mL) or SCF (100 ng/mL). Antibodies raised against the proteins are indicated on the left of the panel. P-Stat5 and P-Kit antibodies detect Stat5 and Kit phosphorylated forms. The blot was probed with an anti-β-actin antibody to visualize the protein loading. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to β actin expression. The fold change in Kit expression between Epo or SCF-cultured cells is indicated at the bottom of each cell line. C: Representative diagram of flow cytometry analysis showing cell surface expression of Kit in 663 and 812 cells cultured with Epo (dotted line) or SCF (black line). Control IgG profile is shown in grey. The table indicates the mean fluorescence intensity (MFI)±SD of positive cells from four independent experiments.</p

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies - Fig 3

<p><b>A Combination treatment of duvelisib plus dexamethasone or ibrutinib leads to sustained inhibition of pAKT</b>. Western blot analysis of DOHH2 (top) and SU-DHL-4 (bottom) treated with duvelisib (1 μM), dexamethasone (500 nM), ibrutinib (100nM) or the combination of duvelisib plus dexamethasone or ibrutinib for 6 hours or 24 hours and stained with pAKT (S473). <b>B. mTOR inhibition can suppress re-phosphorylation of pAKT.</b> SU-DHL-4 cells treated with duvelisib (1 μM), the pan-PI3K inhibitor GDC-0941(500 nM) or the ERK inhibitor SCH-772984 (500 nM) resulted in re-phosphorylation of pAKT (S473) at 24 hours, while combination with the PI3K/mTOR inhibitor PF-04691502 (500 nM) prevented re-phosphorylation of pAKT at 24 hours. <b>C. Re-phosphorylation of pAKT after duvelisib treatment is dependent on mTORC2</b>. DOHH2 (top) or SU-DHL-4 (bottom) treated with duvelisib (1 μM), mTOR1/2 inhibitor AZD-8055 (200 nM) or the mTORC1 inhibitor rapamycin (100 nM) for 1 hour (left) or following a 24 hour treatment with duvelisib (1 μM)(right). <b>D. Duvelisib combinations with dexamethasone and ibrutinib reduce activation of downstream effectors.</b> DOHH2 (top) and SU-DHL-4 (bottom) were treated with duvelisib, dexamethasone and ibrutinib as in Fig 3A. Western blot staining for downstream effectors pPRAS40 (T246), pP70S6K (T389) and pS6 (S235/236). <b>E. Combination of duvelisib with dexamethasone or ibrutinib leads to inhibition of cap-dependent translation</b>. DOHH2 (left) and SU-DHL-4 (right) treated as in Fig 3A and western blot stained for p4EBP1 (S65), c-MYC, peIF4E (s209) and cleaved PARP. The total eIF4E blots demonstrate that equal amounts of protein were loaded per lane.</p

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies - Fig 1

<p><b>A. Growth inhibition (GI₅₀) of duvelisib across the panel of twenty cell lines</b>. The median GI₅₀ across the cell line panel is 0.59 μM. In six cell lines (<b>A & B</b>) with the asterisks (RL, KARPAS-299, RPMI-8226, GRANTA-519, OCI-Ly7, OPM-2), the GI₅₀ failed to reach growth Inhibition levels of greater than fifty percent. <b>B. Growth inhibition (GI₅₀) of duvelisib across the panel of twenty cell lines grouped by tumor subtype</b>. The median GI₅₀ across the cell line panel is 0.59 μM. Cell lines are grouped according to tumor subtypes: Diffuse large B cell lymphoma (DLBCL) activated B cell (ABC) and germinal center B cell (GCB), follicular lymphoma (FL), T cell lymphoma (T cell), mantle cell lymphoma (MCL) and multiple myeloma (MM).</p

The phosphoinositide-3 kinase (PI3K)-δ,γ inhibitor, duvelisib shows preclinical synergy with multiple targeted therapies in hematologic malignancies - Fig 2

<p><b>A. Synergy scores for combination of duvelisib with drugs with different mechanisms of action</b>. Synergy Scores that exceed the mean self-cross plus two times the standard deviation are highlighted in purple. <b>B. Synergy score heat maps for duvelisib combined with dexamethasone, ibrutinib and venetoclax.</b> Representative heat maps for indicated combinations in the TMD-8, SUDHL-4 and DOHH2 cell lines. Scores up to 100 indicate growth inhibition while values >100 indicate cell death.</p