Haematopoiesis is the process through which cellular blood components are produced. It starts during embryonic development to ensure the production of blood cells such as erythrocytes (red cells), leukocytes (white cells), and platelets and continues throughout our lives. All blood cells derive from haematopoietic stem cells located in the bone marrow and, unfortunately, blood cancers may occur during this process. Whether blood cells become inefficient or grow excessively, the outcomes are usually devastating. Dr Chen Zhao (University of Iowa) and Dr Qianze Dong (China Medical University) are exploring the cellular mechanisms of haematopoietic stem cells.

Canonical and Non-Canonical Pathways

Haematopoietic stem/progenitor cells (HSPCs) are highly regulated cells, with the unique ability to self-renew and to differentiate into the different blood cell types. The rate of cell production is controlled by the body’s need. For example, more white cells or platelets are respectively produced to compensate for infection or blood loss. One HPSC undergoes a series of transformations to evolve into a definitive cell type. This is controlled by a multitude of signals coming from the bone marrow microenvironment to the cell DNA and influences the cell differentiation via activation or inactivation of genes. This series of signals is known as the cellular signalling pathways. They can be described as ‘canonical’, referring to traditional pathways specific to cell or tissue or ‘non-canonical’, where pathways diverge from the classical path. The alternative pathways are less studied but now attracting scientific curiosity.

Blood Cells

The Role of the Non-Canonical Pathway in Haematopoiesis

Dr Zhao at the University of Iowa, Dr Qianze Dong at China Medical University, and their colleagues are interested in the well-known NF-κB pathway (nuclear factor kappa-light-chain-enhancer of activated B cells). It includes a collection of different proteins modulating physiological processes such as immune responses, cell proliferation or apoptosis (cell death). NF-κB is expressed in most cells and controls the expression of numerous genes. However, the role of NF-κB signalling to regulate HSPCs is still unclear.

NF-κB is tightly regulated by intermediate signalling molecules, such as the NF-κB inducing kinase (NIK), a crucial kinase of the non-canonical pathways. A kinase is an enzyme allowing the transfer of a phosphate group between two molecules, allowing the signal to spread into the cell from the membrane to the nucleus where the DNA is located. Previous research has established that inactivating NIK impairs the ability of HSPCs to self-renew.

Whilst the role of the canonical NF-κB pathway is already well described, Dr Zhao and his colleagues are interested in the alternative or non-canonical NF-κB pathway. Its role in normal and pathologic haematopoiesis has been overlooked over the last decade. Dr Zhao previously made the novel observation that non-canonical NF-κB signalling supports HSPC self-renewal and preserves the stem cell pool. Starting from this discovery, Dr Zhao progressed the idea that constant activation of the non-canonical pathway via the permanent activation of NIK could influence haematopoiesis. To test this, Dr Zhao and his team developed a new genetically modified mouse model, where NIK is constantly activated either specifically in the HSPCs or in the whole body.

The mutant mice, NIK-inactivated in HSPC, rapidly showed growth defects with reduced body and organ sizes with a life expectancy of 7 days. This was accompanied by reduced levels of erythrocytes, leukocytes, or platelets in the blood. In contrast to Dr Zhao and his team’s expectations, a permanent activation of NIK appeared to be detrimental to normal haematopoiesis. They further demonstrated that the activation of NIK compromises the health of HSPCs, increasing inflammation and cell death. These findings highlight the opposite effects of the canonical and non-canonical NF-κB pathways and the role of stable NIK to maintain the healthy production of blood components.

The Role of Non-Canonical Pathways in Acute Myeloid Leukaemia. Credit Chen Zhao.

The Role of the Non-Canonical Pathway in Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is an aggressive form of blood cell cancer, characterised by the rapid growth of abnormal blood cells, preventing healthy cells from being efficient. It is caused by a small pool of malignant cells, able to self-renew, called leukaemia stem cells (LSCs). Most current treatments involve chemotherapy but understanding the LSC signalling pathways is a promising therapeutic approach aiming to stop them from proliferating. Canonical NF-κB signalling is activated in LSCs and is associated with higher resistance to chemotherapy. It had previously been demonstrated that suppression of the canonical pathway, combined with other therapies, helps to slow down AML development.

Once again, Dr Zhao and his colleagues thought outside the box and explored the role of the non-canonical NF-κB pathway in LSCs. If permanently activating NIK in a healthy context might be detrimental, it might, however, promote the self-renewal of stem cells in pathological conditions such as AML. They used the same mouse model, NIK-activated in the whole body (stabilisation), in combination with the well-characterised MLL-AF9, a genetic recombination mimicking the effects of DNA modifications that frequently result in the formation of human cancer. In this case, MLL-AF9 was used to induce AML.

This study showed that the stabilisation of NIK activated the non-canonical and repressed the canonical pathway. Activation of the NF-κB non-canonical pathway upregulates Dnmt3a and downregulates Mef2c, two genes respectively suppressing and promoting AML. The hypothesis behind this mechanism is that the non-canonical pathway negatively controls the gene responsible for the LSC self-renewal and positively control the genes involved in LSC suppression. Whilst further studies are required to pinpoint the exact mechanisms, this study highlights the ability of NIK stabilisation to suppress AML development and lays the foundation for therapeutic progress.

Identification of Verteporfin to Suppress AML

As the overactivation of NIK impairs the self-renewal of healthy HSPCs, a key challenge is to stabilise NIK only in LSCs. Dr Zhao and his colleagues observed that activating the non-canonical NF-κB pathway via NIK modulates the expression of different genes responsible for the survival and the death of LSCs. A promising therapeutic approach is to identify a drug that targets these specific genes.

The Connectivity Map (CMAP) database is a very useful tool in drug development. It includes a large collection of gene expression profiles from cultured human cells stimulated with various chemicals. The database allows scientists to identify any component responsible for the modulation of genes of interests. Dr Zhao used CMAP, targeting LSC genes, and identified verteporfin as a potential candidate for the treatment of AML. Verteporfin is a well-known treatment that suppresses abnormal vessels responsible for blurred vision in macular degeneration. It was also proven to inhibit tumour growth in various cancer models such as acute lymphoblastic leukaemia, with minimal effects on normal haematopoiesis. Testing verteporfin in vitro was efficient to reduced LSC proliferation and delayed AML development.

A New Model of Lymphoma. Credit Chen Zhao.

A New In Vivo Model to Study Lymphomas and Lymphoma to Leukaemia Conversion

B cell lymphomas are a type of blood cancers affecting the B cells in the lymph nodes. It is sometimes associated with the apparition of myeloid tumours such as AML. The rare conversion of lymphoma to AML is well recognised but the underlying molecular mechanisms are still poorly understood. This is mainly due to the lack of efficient in vivo models. In a recent study, Dr Zhao explained ‘Because most patients in whom B-cell lymphoma undergoes conversion to myeloid tumour have a poor prognosis as a result of diagnostic difficulties and lack of standard treatment, it is important to elucidate the biological underpinning of the B-to-myeloid switch and develop new approaches to treat and prevent these uncommon but usually fatal neoplasms.’

Previous studies had already demonstrated that activation of NF-κB or Notch (neurogenic locus notch homolog protein) in B-cells is not sufficient to induce B cell Lymphoma. This is why Dr Zhao and Dr Dong proposed a new mouse model with concurrent activation of NF-kB and Notch signalling in committed B cells. This model is particularly interesting and adequate as coactivation of NF-kB/Notch signalling in B cells significantly accelerates lymphoma development in mice and has the ability to convert to myeloid lineage, this is the lymphoma to leukaemia conversion similarly observed in patients

After validating the model in vivo, Dr Dong transplanted B cells activated for NF-κB and Notch into healthy mice. Lymphoma to leukaemia conversion occurred in 15% of the mice suggesting that simultaneous activation of both pathways is responsible for B cell lymphoma and conversion to AML. ‘Targeting Notch/NF-kB pathways may not only facilitate lymphoma treatment, but also prevent B-myeloid conversion’ explains Dr Dong.

Further experiments revealed that DNA methylation, a process by which methyl groups are added to the DNA strands, also plays an important role during the lymphoma-to-leukaemia conversion. DNA methylation regulates the accessibility of the DNA, influencing the gene expression. A drug suppressing DNA methylation effectively reduced converted cells but the underlying mechanisms of this process remain to be clarified.

Dr Zhao’s findings represent major scientific breakthroughs. The identification of the role of the non-canonical pathway progressively opens doors to understand the mechanisms of health and poor haematopoiesis in different cancer models such as acute myeloid leukaemia or B cell lymphomas. As such, this early-stage laboratory work holds great promise for the development of alternative anti-cancerous treatments.

Reference
https://doi.org/10.33548/SCIENTIA507

Meet the researchers

Dr Chen Zhao
Associate Professor
University of Iowa Hospitals and Clinics
Iowa City, IA
USA

Dr Chen Zhao graduated medical school in 1993 from the China Medical University of Shenyang and completed his PhD in 2002 at Keio University School of Medicine, Tokyo. In 2004, Dr Zhao moved to the USA where he continues his academic path. Having completed a range of clinical and research positions, Dr Zhao is now a tenured Associate Professor at the University of Iowa. With a number of publications in the highest ranking journals including Nature, Dr Zhao has an impressive scientific track record.

 

CONTACT

E: chen-zhao@uiowa.edu

W: https://zhao.lab.uiowa.edu/

 

FUNDING

Veterans Health Administration Merit Review Program

National Institutes of Health

University of Iowa and Mayo Clinic SPORE Developmental Research Program Award

American Cancer Society (ACS) Seed Grant of Iowa/American Cancer Society     

 

Dr Qianze Dong
Professor of Pathology
China Medical University and the First Affiliated Hospital of China Medical University
China

In 2009, Dr Qianze Dong completed his medical degree at the China Medical University where he also begun his research career with a PhD in the Department of Pathology. In 2011, Dr Dong obtained title of Assistant Professor followed by Associate Professor in 2013. He is now a Professor in the Department of Pathology, China Medical University and the First Affiliated Hospital of China Medical University. Since 2017, Dr Dong has been a visiting scholar at Dr Zhao’s laboratory at the University of Iowa School of Medicine, USA. Together, they are together unravelling the secrets of the cellular signaling pathways involved in haematopoiesis and related blood cancers.

 

CONTACT

E: dongqianze@mail.cmu.edu.cn

 

FUNDING

National Natural Science Foundation of China

 

FURTHER READING

Y Xiu, Q Dong, L Fu, et al, Coactivation of NF-κB and notch signaling is sufficient to induce B-cell transformation and enables B-myeloid conversion, Blood, 2020, 135(2), 108–120.

Y Xiu, Q Dong, Q Li, et al, Stabilization of NF-κB-inducing kinase suppresses MLL-AF9-induced acute myeloid leukemia, Cell Reports, 2018, 22(2), 350–358.

Y Xiu, WY Xue, A Lambertz, et al, Constitutive activation of NIK impairs the self-renewal of hematopoietic stem/progenitor cells and induces bone marrow failure, Stem Cells, 2017, 35(3), 777–789.

 

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