Inhibition Of Stem Cell Proliferation Research Articles

Abstract 2695: Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors

Abstract NOTCH signalling is a key development pathway whose aberrant activation plays an onco-driver role in human cancers. In human tumors the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss-of-function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blocking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumors harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumors. On the other hand, GSIs act as pan-NOTCH inhibitors and thereby are able to target human tumors positive for NOTCH1-NOTCH4 receptors. However, clinical development of these pan-NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in intestine, where NOTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effect on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, cMYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre-requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1st and 2nd generation NOTCH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan-NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan-NOTCH inhibitors that blocks signalling by directly targeting the NOTCH transcription complex. Here we present further in vivo characterization of the development candidate CB-103. A comprehensive in vitro study demonstrated that CB-103 acts as a pan-NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB-103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB-103 fully engages the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia. Citation Format: Rajwinder Lehal, Jelena Zaric, Michele Vigolo, Charlotte Urech, Maximilien Murone, Freddy Radtke. Direct targeting of NOTCH transcription complex by a novel small molecule CB-103 circumvents dose-limiting toxicities associated with pan-NOTCH inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2695.

Thymosin β as an Actin-binding Protein with a Variety of Functions.

According to current data, the thymosin β family is composed of 20 short (40-44 amino acid) peptides, but in a healthy human body only 2 are expressed - thymosin β4 and β10. Their most characteristic feature is the ability to form a complex with monomeric actin, thereby preventing polymerization into a filamentous form, hence the name Actin-Binding Protein (ABP). These peptides play numerous different functions. Among others, they affect the processes of carcinogenesis, differentiation and angiogenesis, influence metalloproteinase activity and accelerate wound healing. Moreover, significant biological activity has also been displayed by Tβ4 derived peptides: Ac-SDKP, the N-terminal fragment which is involved, inter alia, in stimulating angiogenesis and the inhibition of stem cell proliferation and Tβ4 sulfoxide, an oxidation product of one of the peptide methionine by hydrogen peroxide, which inhibit the development of inflammation. The properties of these peptides have potential applications in cardiovascular medicine, dermatology, ophthalmology and other medical areas.

Induction of Toll-Like Receptor 3-Mediated Immunity during Gestation Inhibits Cortical Neurogenesis and Causes Behavioral Disturbances

Maternal infection during pregnancy with a wide range of RNA and DNA viruses is associated with increased risk for schizophrenia and autism in their offspring. A common feature in these exposures is that virus replication induces innate immunity through interaction with Toll-like receptors (TLRs). We employed a mouse model wherein pregnant mice were exposed to polyinosinic-polycytidylic acid [poly(I ⋅ C)], a synthetic, double-stranded RNA molecular mimic of replicating virus. Poly(I ⋅ C) inhibited embryonic neuronal stem cell replication and population of the superficial layers of the neocortex by neurons. Poly(I ⋅ C) also led to impaired neonatal locomotor development and abnormal sensorimotor gating responses in adult offspring. Using Toll-like receptor 3 (TLR3)-deficient mice, we established that these effects were dependent on TLR3. Inhibition of stem cell proliferation was also abrogated by pretreatment with the nonsteroidal anti-inflammatory drug (NSAID) carprofen, a cyclooxygenase (COX) inhibitor. Our findings provide insights into mechanisms by which maternal infection can induce subtle neuropathology and behavioral dysfunction, and they may suggest strategies for reducing the risk of neuropsychiatric disorders subsequent to prenatal exposures to pathogens and other triggers of innate immunity.

Curcumin Functions as a Positive Regulator of miRNA Processing & a Negative Regulator of Cancer Stem Cell Proliferation

AbstractIdentifying agents that inhibit the proliferation of cancer stem cells is of great importance in cancer biology. Curcumin has been shown to function as an anti-cancer agent. One of the tumor suppressors that appear to be induced in response to curcumin treatment is p53. A recent study suggests that curcumin increases the expression of miRNAs, such as miR-22, miR-181a, b, c, miR-34, miR-103, and miR-21. Intriguingly, miR-34, 181b, c, miR-103, miR-21, and miR-24 have been identified as transcriptional targets of the tumor suppressor p53. This data suggests a possibility that curcumin, by inducing the expression of p53, it could increase the expression of these microRNAs. A number of groups have shown that c-Myc, Sox-2, Klf-4, Oct-4, Sox-2, Nanog, and Lin28 are required for reprogramming of differentiated cells into pluripotent stem cells and for cancer stem cell proliferation. Interestingly, miR-21 has recently been shown to represses stem cell factors such as Oct4, Nanog, sox2, and c-Myc, indicating that curcumin by increasing the expression of miR-21, it could inhibit cancer stem cell proliferation. In addition, curcumin induced p53 may result in increased expression of its target, miR-145. Interestingly, it has recently been shown that miR-145 suppresses the expression of c-myc, klf-4, Oct-4, and Sox-2 in human embryonic stem cells (hESCs) and thereby promotes the differentiation of hESC. This data suggests that p53-dependent miR-145 expression will result in down regulation of Oct-4, Klf-4, Sox-2, Nanog, and c-myc and inhibition of stem cell proliferation. Further, curcumin induced miR-22 appears to inhibit the expression of estrogen receptor α. Remarkably, it has recently been shown that estrogen receptor α inhibits the processing of several microRNAs, including the tumor suppressor miR-145. This data suggests that curcumin, by inhibiting the expression of estrogen receptor α. through its target miR-22, it could increase the processing of miR-145 and down regulate the expression of its target genes (Oct-4, Klf-4, Sox-2, Nanog, and c-myc). Thereby, curcumin could function as a positive regulator of miRNA processing and a negative regulator of cancer stem cell proliferation.

Mechanisms of thrombocytopenia induced by interferon therapy for chronic hepatitis B.

To clarify the mechanisms of thrombocytopenia observed in patients with chronic hepatitis B treated with interferon. We studied six patients with chronic active hepatitis B who received intramuscular injections of natural interferon-alpha (3 or 5 million IU/ day) for 4 weeks. Peripheral blood platelet counts, bone marrow findings, and platelet kinetics, determined using 111In-labeled platelets, were analyzed. Platelets decreased significantly 1 week after the beginning of treatment and remained decreased until the completion of treatment. The number of nucleated cells and megakaryocytes in bone marrow decreased in three of five patients studied during treatment. The kinetic study showed platelet survival time to be 8.1 +/- 1.3 days (range, 5.8-10.0). One day after platelet injection, platelets accumulated predominantly in the splenic area in all patients, whereas hepatic accumulation was predominant 7 days after injection in three of the six patients. Thrombocytopenia during interferon treatment arises from the inhibition of stem cell proliferation and differentiation in the bone marrow and from the capture of platelets by the liver.

Inhibition of enriched stem cells in vivo and in vitro by the hemoregulatory peptide SK&F108636.

Replacement of the labile sulfhydryl group (-SH) of the hemoregulatory peptide monomer pyroGluGluAspCysLys (HP5b) with an isosteric methylene group yields a chemically stable compound, SK&F108636. In this study, we describe the effects of SK&F108636 on highly enriched Lin-Sca1+ hematopoietic stem cells. SK&F108636 significantly reduced the fraction of cycling progenitor cells, granulocyte macrophage colony-forming cells (GM-CFC), in vitro and in vivo. There was no effect on GM-CFC or Mix-CFC colony formation. SK&F108636 significantly inhibited proliferation of high proliferative potential (HPP)-CFC in semisolid agar cultures stimulated by stem cell factor + interleukin 3 (IL-3) + IL-1, but had no effect in cultures stimulated with M-CSF + IL-3 + IL-1. SK&F108636 was shown to act directly on the stem cells since SK&F108636 inhibited proliferation of Lin-Sca1+ cells in single cell assays. Administration of SK&F108636 to lethally irradiated mice transplanted with 2000 Lin-Sca1+ cells significantly inhibited proliferation/differentiation of cells developing into colony forming units-spleen (CFU-S) (preCFU-S) and the reconstitution of HPP-CFC and GM-CFC. There was no effect of SK&F108636 on CFU-S colony formation or mature cell regeneration in bone marrow, spleen and blood. Hence, the hemoregulatory peptide monomer SK&F108636 is a potent primitive stem cell inhibitor in vivo and in vitro. Inhibition of stem cell proliferation by small specific inhibitors may protect hematopoiesis from myelotoxic side effects during chemotherapy treatment.

Recovery of hematopoiesis after blood-group-incompatible bone marrow transplantation with red-blood-cell-depleted grafts.

Severe hemolytic transfusion reactions may complicate major blood-group-incompatible bone marrow transplantations (BMT). Probably the most appropriate way to avoid this complication is removal of the incompatible red blood cells (RBC) from the bone marrow graft. In this report we describe a method to eliminate incompatible RBCs that is based on repeated dilution of the graft with donor-and-recipient--compatible third-party erythrocytes. Four patients with ABO incompatibility and one patient with Rh-C incompatibility were transplanted using this technique. After the procedure 86 +/- 8% (mean +/- SD) of the nucleated cells, 95 +/- 14% of CFU-GM, 88 +/- 14% of BFU-e, and 103 +/- 30% of CFU-e were recovered, but only +/- 1% of the incompatible RBCs was left in the transfusate (1-3 ml). No signs of hemolysis were observed. All patients engrafted promptly. The patients with low titers of antibody had normal reticulocyte recoveries. Maturation of erythropoiesis was suppressed only when high titers of antibody were present. However, despite high antibody titers, erythroid progenitor cells (CFU-GEMM, BFU-e, and CFU-e) could be cultured from the bone marrow of the recipient after BMT. Thus, anti-A and anti-Rh-C antibodies give little, if any, inhibition of stem-cell proliferation. The method described to remove incompatible RBCs appears to be simple, safe, and--in most cases--sufficient.

The Regulation of Hemopoiesis in Long-Term Bone Marrow Cultures. II. Stimulation and Inhibition of Stem Cell Proliferation

The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long-term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8-10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).

The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation

The isolation of a DNA synthesis inhibitor (NBME fraction IV) and stimulator (RBME fraction III) specific for the hemopoietic stem cell (CFU-s) from freshly isolated normal adult and regenerating murine bone marrow, respectively, has been well documented. We have utilized long- term liquid bone marrow cultures in a further analysis of the role of these factors in the regulation of CFU-s proliferation. Our results show that shortly after feeding, at a time when the cultured CFU-s are actively proliferating, high levels of the hemopoietic stem cell proliferation stimulator fraction III can be isolated from the culture medium. In contrast, the presence of essentially noncycling CFU-s found in cultures fed 8–10 days previously correlates with high levels of the hemopoietic stem cell inhibitor fraction IV. These results suggest that a certain balance between these factors determines CFU-s proliferation in the long-term cultures. In support of this, DNA synthesis in actively cycling CFU-s in the long-term cultures is inhibited for at least 3 days by the addition of excess NBME fraction IV (inhibitor). Furthermore, DNA synthesis in noncycling cultured CFU-s is stimulated for at least 5 days by the addition of RBME fraction III (stimulator).