Estrogen Receptors participate in both genomic and non‐genomic signaling pathways that affect breast cancer cell growth and proliferation. The growth‐enhancing actions of estrogen (E2) in MCF‐7 ...breast cancer cells are mediated through Estrogen Receptor alpha (ERα), activating a kinase pathway involving CaM KK, CaM KI, and ERK. Conversely, overexpression of Estrogen Receptor beta (ERβ) has been shown to restrict the transcriptional activity of ERα and inhibit breast cancer cell growth. Our goal was to explore the roles of ERα and ERβ in ERK activation and cell growth in MCF‐7 and MDA‐MB‐231 breast cancer cells. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERα antagonist MPP and siRNA directed against ERα. Inhibition of ERβ with its antagonist PHTPP did not reduce ERK activity following stimulation with E2. Interestingly, pretreatment of MCF‐7 cells with the ERβ agonist, FERb, prevented the activation of ERK by E2. Treatment of MCF‐7 cells with E2 stimulated an increase in cell growth that was blocked by pretreatment with MPP, but not by pretreatment with PHTPP. In both MCF‐7 and MDA‐MB‐231 cells, stimulation with FERb reduced cell growth slightly below control levels. Treatment of MDA‐MB‐231 cells with FERb rapidly increased ERK phosphorylation and this effect was blocked by pretreatment with PHTPP. Our data suggest ERβ differentially regulates ERK in MCF‐7 and MDA‐MB‐231 cells. While activation of ERβ inhibits the growth of both cell lines, ERβ prevents ERK phosphorylation in MCF‐7 cells, but enhances ERK phosphorylation in MDA‐MB‐231 cells.
Grant Funding Source: Supported by M.J.Murdock Charitable Trust #2011267
To identify new therapeutic targets in acute myeloid leukemia (AML), we performed small-molecule and small-interfering RNA (siRNA) screens of primary AML patient samples. In 23% of samples, we found ...sensitivity to inhibition of colony-stimulating factor 1 (CSF1) receptor (CSF1R), a receptor tyrosine kinase responsible for survival, proliferation, and differentiation of myeloid-lineage cells. Sensitivity to CSF1R inhibitor GW-2580 was found preferentially in de novo and favorable-risk patients, and resistance to GW-2580 was associated with reduced overall survival. Using flow cytometry, we discovered that CSF1R is not expressed on the majority of leukemic blasts but instead on a subpopulation of supportive cells. Comparison of CSF1R-expressing cells in AML vs healthy donors by mass cytometry revealed expression of unique cell-surface markers. The quantity of CSF1R-expressing cells correlated with GW-2580 sensitivity. Exposure of primary AML patient samples to a panel of recombinant cytokines revealed that CSF1R inhibitor sensitivity correlated with a growth response to CSF1R ligand, CSF1, and other cytokines, including hepatocyte growth factor (HGF). The addition of CSF1 increased the secretion of HGF and other cytokines in conditioned media from AML patient samples, whereas adding GW-2580 reduced their secretion. In untreated cells, HGF levels correlated significantly with GW-2580 sensitivity. Finally, recombinant HGF and HS-5–conditioned media rescued cell viability after GW-2580 treatment in AML patient samples. Our results suggest that CSF1R-expressing cells support the bulk leukemia population through the secretion of HGF and other cytokines. This study identifies CSF1R as a novel therapeutic target of AML and provides a mechanism of paracrine cytokine/growth factor signaling in this disease.
•CSF1R inhibition reduces cell viability in >20% of AML patient samples and is expressed on a subpopulation of supportive cells.•CSF1R activation stimulates paracrine cytokine secretion (eg, HGF), suggesting that CSF1R is a novel target of AML support cells.
Display omitted
Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of ...mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.
Translating the genetic and epigenetic heterogeneity underlying human cancers into therapeutic strategies is an ongoing challenge. Large-scale sequencing efforts have uncovered a spectrum of ...mutations in many hematologic malignancies, including acute myeloid leukemia (AML), suggesting that combinations of agents will be required to treat these diseases effectively. Combinatorial approaches will also be critical for combating the emergence of genetically heterogeneous subclones, rescue signals in the microenvironment, and tumor-intrinsic feedback pathways that all contribute to disease relapse. To identify novel and effective drug combinations, we performed ex vivo sensitivity profiling of 122 primary patient samples from a variety of hematologic malignancies against a panel of 48 drug combinations. The combinations were designed as drug pairs that target nonoverlapping biological pathways and comprise drugs from different classes, preferably with Food and Drug Administration approval. A combination ratio (CR) was derived for each drug pair, and CRs were evaluated with respect to diagnostic categories as well as against genetic, cytogenetic, and cellular phenotypes of specimens from the two largest disease categories: AML and chronic lymphocytic leukemia (CLL). Nearly all tested combinations involving a BCL2 inhibitor showed additional benefit in patients with myeloid malignancies, whereas select combinations involving PI3K, CSF1R, or bromodomain inhibitors showed preferential benefit in lymphoid malignancies. Expanded analyses of patients with AML and CLL revealed specific patterns of ex vivo drug combination efficacy that were associated with select genetic, cytogenetic, and phenotypic disease subsets, warranting further evaluation. These findings highlight the heuristic value of an integrated functional genomic approach to the identification of novel treatment strategies for hematologic malignancies.
Abstract only
The rapid signaling effects of the hormone estrogen (E2) are mediated through estrogen receptor alpha (ERα) that activates a Ca
2+
/calmodulin signaling pathway involving CaM KK, CaM ...KI, and ERK. Additionally, expression of transient receptor potential (TRP) channels, which are Ca
2+
channels, may correlate with breast cancer cell growth. In this family of channels, TRPC6 has been shown to be expressed in MCF‐7 breast cancer cells and involved in ERK activation. Since both ERα and TRPC6 increase intracellular calcium levels, ERK activation, and cell growth our goal was to examine if ERα cooperates with TRPC6 to increase CaM Kinase and ERK phosphorylation as well as MCF‐7 cell growth. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERa antagonist MPP. Treatment of MCF‐7 cells with E2 stimulated an increase in MCF‐7 cell growth that was blocked by pretreatment with MPP, but not by pretreatment with the ERb antagonist PHTPP. Interestingly, treatment of cells with the TRP channel inhibitor APB blocked E2 activation of ERK and cell growth. Immunoprecipitation (IP) of endogenous ERa pulled down TRPC6 as did the reciprocal IP from MCF‐7 cells. Confocal microscopy also revealed co‐localization of the proteins in MCF‐7 cells. Our data suggest E2 rapidly activates CaM KI and ERK via ERa and TRP channels and ERa may associate with TRPC6 to regulate breast cancer cell growth.
Support or Funding Information
The Paul K. and Evalyn E. C. Richter Memorial Fund.
This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.
Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous ...myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.
•First comprehensive genomic and transcriptomic profiling of CNL, aCML, and MDS/MPN-U.•Diagnoses represent a continuum of related diseases rather than discrete diagnostic entities.
Display omitted
Abstract only
Estrogen (E2) signaling significantly affects breast tumorigenesis by enhancing cell growth and preventing apoptosis. The actions of estrogen in MCF‐7 breast cancer cells are mediated ...through a kinase pathway involving CaM KK, CaM KI, and ERK. Current research is examining the involvement of the Estrogen Receptor (ER) alpha (α) and beta (β) as well as G‐Protein Coupled Receptor GPR30 in cell growth and proliferation. ERα is suggested to be responsible for ERK and perhaps CaM Kinase activation. Our goal was to evaluate if ERα, rather than GPR30 or ERβ, mediates CaM KI and ERK activation upon treatment of MCF‐7 breast cancer cells with E2. E2 treatment of MCF‐7 cells led to a significant rise in CaM KI and ERK activation. This effect was blocked by pretreatment with the ERα antagonist MPP; however MPP did not block EGF stimulation of ERK. We then explored the role of ERβ in E2‐mediated ERK activation by pretreating cells with the ERβ inhibitor PHTPP. PHTPP did not reduce ERK phosphorylation, suggesting ERβ is not involved in activation of ERK. MCF‐7 cells were transfected with siRNA against ERα to further examine the role of ERα in ERK regulation. Knockout of ERα in MCF‐7 cells subsequently treated with E2 showed a significant decrease in ERK phosphorylation. To ensure the specificity of the siRNA for ERα, transfected cells were stimulated with EGF. ERK phosphorylation following EGF treatment was not inhibited in ERα‐knockout cells. Taken together our data suggests that ERα is capable of rapidly activating both CaM KI and ERK in MCF‐7 cells.
In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive ...monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.
Introduction: CPX-351 (Vyxeos), a liposomal combination of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio, has recently been shown to be significantly more efficacious ...than the standard of care, 7+3 administration regimen of free (non-liposomal) cytarabine + daunorubicin in treating elderly high-risk AML patients, including patients with the FLT3-ITD mutation. We previously exposed AML blasts from primary patient samples to CPX-351 ex vivo and found that samples harboring FLT3-ITD were significantly more sensitive to CPX-351 and also demonstrated enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in upregulated liposome uptake pathways, leading to increased cell death and overall sensitivity to CPX-351. Furthermore, we wanted to examine the effect of combining CPX with existing FLT3 inhibitors (e.g. quizartinib or midostaurin).
Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains mutant, activated FLT3) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We measured cell viability and intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry and using a fluorescent plate reader. Additionally, we performed synergy analyses on the cytotoxic activity in these cells, adding CPX-351 and FLT3 inhibitors in combination at different exposure sequences followed by measurement of cell viability and daunorubicin fluorescence.
Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351, and exhibited increased drug uptake, compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, indicating that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, in synergy studies we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses.
Conclusions: These results provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351, which is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling.
Tardi:Jazz Pharmaceuticals: Employment. Mayer:Jazz Pharmaceuticals: Employment.
PDF
