Small molecule mediated targeting of haematopoietic stem/progenitor cell and leukaemic stem cell function

Haematopoietic stem cells (HSC) are a rare population of cells that have the ability to self-renew and differentiate giving rise to various blood lineages, thereby reconstituting the whole haematopoietic system. This is an essential characteristic, exploited in bone marrow transplantation therapy in response to myeloablative treatment. Due to their rarity, the lack of sufficient HSC numbers for transplantation has proved to be a major clinical issue. Separately, in the development of leukaemia, acquired mutations in HSCs give rise to malignant cells. These cells, like HSCs, have the ability to self-renew and differentiate forming immature blasts and are termed cancer (leukaemic) stem cells. They are thought to remain in a quiescent state and are therefore not targeted by standard chemotherapy, inducing relapse in haematopoietic malignancies. In this study, a cross species stem cell based screen was conducted on a 12,000 small molecule library across a range of adult and embryonic tissue types with a view to identifying compounds that would (i) expand HSCs ex vivo and in vivo for transplantation and (ii) eradicate cancer stem cells in leukaemia. A number of small molecules were identified as lead compounds and were assessed in our investigation. We found that Yohimbine, an alpha-2 adrenergic receptor (adra-2) antagonist, and Oxa-22, cis-2-Methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide (M3 Muscarinic acetylcholine receptor agonist) elicited a 2- and 1.5- fold increase in HSC frequency (respectively) in vivo. Further competitive transplantation studies showed that Yohimbine and Oxa-22 treated cells also enhanced the reconstitution of B cells and T cells respectively. In parallel, we also assessed Oxa-22 and a third compound, Phthalylsulfathiazole- an antibacterial sulphonamide, in the leukaemic setting to ascertain whether compounds could target leukaemic stem cells (LSCs). We found that these compounds promoted proliferation in acute myeloid leukaemia (AML) cell lines. Furthermore, when Oxa-22 and Phthalylsulfathiazole were administered in vivo models of AML, they accelerated disease progression by increasing the number of LSCs. Collectively, these results show that using small molecules we can target neuronal related pathways to enhance HSC number and function. Further investigation is required to elucidate the exact mechanisms of the compounds however, these data may prove to be influential in directing new methods of stem cell expansion for transplantation therapies. Small molecules targeting neuronal or antibacterial related pathways were also found to target malignant LSCs and alter their behaviour. By driving LSCs out of their dormant state, these small molecules may pave the way for potential targeting of LSCs in conjuction with standard current chemotherapies that incorporate and kill proliferating cancer cells

Zeb1 modulates hematopoietic stem cell fates required for suppressing acute myeloid leukemia

Zeb1, a zinc finger E-box binding homeobox epithelial-mesenchymal (EMT) transcription factor, confers properties of ‘stemness’, such as self-renewal, in cancer. Yet little is known about the function of Zeb1 in adult stem cells. Here, we used the hematopoietic system, as a well-established paradigm of stem cell biology, to evaluate Zeb1 mediated regulation of adult stem cells. We employed a conditional genetic approach using the Mx1-Cre system to specifically knockout (KO) Zeb1 in adult hematopoietic stem cells (HSCs) and their downstream progeny. Acute genetic deletion of Zeb1 led to rapid onset thymic atrophy and apoptosis driven loss of thymocytes and T cells. A profound cell-autonomous self-renewal defect and multi-lineage differentiation block was observed in Zeb1 KO HSCs. Loss of Zeb1 in HSCs activated transcriptional programs of deregulated HSC maintenance and multi-lineage differentiation genes, and of cell polarity, consisting of cytoskeleton, lipid metabolism/lipid membrane and cell adhesion related genes. Notably, Epithelial cell adhesion molecule (EpCAM) expression was prodigiously upregulated in Zeb1 KO HSCs, which correlated with enhanced cell survival, diminished mitochondrial metabolism, ribosome biogenesis, and differentiation capacity and an activated transcriptomic signature associated with acute myeloid leukemia (AML) signaling. ZEB1 expression was downregulated in AML patients and Zeb1 KO in the malignant counterparts of HSCs - leukemic stem cells (LSCs) - accelerated MLL-AF9 and Meis1a/Hoxa9-driven AML progression, implicating Zeb1 as a tumor suppressor in AML LSCs. Thus, Zeb1 acts as a transcriptional regulator in hematopoiesis, critically co-ordinating HSC self-renewal, apoptotic and multi-lineage differentiation fates required to suppress leukemic potential in AML

Gata2 as a crucial regulator of stem cells in adult hematopoiesis and acute myeloid leukemia

Subversion of transcription factor (TF) activity in hematopoietic stem/progenitor cells (HSPCs) leads to the development of therapy-resistant leukemic stem cells (LSCs) that drive fulminant acute myeloid leukemia (AML). Using a conditional mouse model where zinc-finger TF Gata2 was deleted specifically in hematopoietic cells, we show that knockout of Gata2 leads to rapid and complete cell-autonomous loss of adult hematopoietic stem cells. By using short hairpin RNAi to target GATA2, we also identify a requirement for GATA2 in human HSPCs. In Meis1a/Hoxa9-driven AML, deletion of Gata2 impedes maintenance and self-renewal of LSCs. Ablation of Gata2 enforces an LSC-specific program of enhanced apoptosis, exemplified by attenuation of anti-apoptotic factor BCL2, and re-instigation of myeloid differentiation––which is characteristically blocked in AML. Thus, GATA2 acts as a critical regulator of normal and leukemic stem cells and mediates transcriptional networks that may be exploited therapeutically to target key facets of LSC behavior in AML

The CDK4/6 Inhibitor Palbociclib Inhibits Estrogen-Positive and Triple Negative Breast Cancer Bone Metastasis In Vivo

CDK 4/6 inhibitors have demonstrated significant improved survival for patients with estrogen receptor (ER) positive breast cancer (BC). However, the ability of these promising agents to inhibit bone metastasis from either ER+ve or triple negative BC (TNBC) remains to be established. We therefore investigated the effects of the CDK 4/6 inhibitor, palbociclib, using in vivo models of breast cancer bone metastasis. In an ER+ve T47D model of spontaneous breast cancer metastasis from the mammary fat pad to bone, primary tumour growth and the number of hind limb skeletal tumours were significantly lower in palbociclib treated animals compared to vehicle controls. In the TNBC MDA-MB-231 model of metastatic outgrowth in bone (intracardiac route), continuous palbociclib treatment significantly inhibited tumour growth in bone compared to vehicle. When a 7-day break was introduced after 28 days (mimicking the clinical schedule), tumour growth resumed and was not inhibited by a second cycle of palbociclib, either alone or when combined with the bone-targeted agent, zoledronic acid (Zol), or a CDK7 inhibitor. Downstream phosphoprotein analysis of the MAPK pathway identified a number of phosphoproteins, such as p38, that may contribute to drug-insensitive tumour growth. These data encourage further investigation of targeting alternative pathways in CDK 4/6-insensitive tumour growth