Catriona H.M. Jamieson, MD PhD | Professor of Medicine Director, Sanford Stem Cell Clinical Center Deputy Director Moores Cancer Center University of California, San Diego

Background

“Slowly, his eyes adjust to the light of the sun. First he can see only shadows. Gradually he can see the reflections of people and things in water and then later see the people and things themselves. Eventually, he is able to look at the stars and moon at night until finally he can look upon the sun itself”

Allegory of the Cave, Republic, Plato (516a)

In the Allegory of the Cave, Plato describes how prisoners in a cave see only the shadows on the wall compared with the freed prisoner who in the light of day can see things themselves (Fig. 1).¹

During a turbulent time in history when the threat of nuclear war was looming, Till and McCulloch discovered a functional rather than a phenotypic shadow of hematopoiesis. By performing quantitative transplantation assays in irradiated mice, they identified a bone marrow-resident dormant cell with multi-lineage differentiation, homing and self-renewal potential that could resist radiation exposure – the hematopoietic stem cell (HSC).^2,3

Subsequently, Spangrude, Heimfeld and Weissman used fluorescence activated cell sorting (FACS) analysis to prospectively purify and characterize the cell surface markers that defined mouse HSCs whereby 30 of the cells could rescue 50 percent of irradiated mice.⁴ These studies were followed in close succession by the identification of human hematopoietic stem cell populations.⁵ The ontogeny of both mouse and human HSCs was further defined by Mikkola and Orkin and refined by Coller, Frenette, Morrison, Suda and Trumpp who underscored the importance of quiescence for HSC maintenance in bone marrow niches.^6,7

As a counterbalance to normal hematopoiesis, seminal research performed by Nowell, Hungerford, Rowley, Witte, Daley, Van Etten and Baltimore demonstrated that a single fusion gene, BCR-ABL, was necessary and sufficient to initiate chronic phase chronic myeloid leukemia (CML), thereby setting the stage for molecularly targeted therapeutic strategies that enabled operational cures albeit with a high risk of relapse following discontinuation of therapy.^3,8 This could be explained at least in part by the discovery by Holyoake and Eaves in mice and Jamieson and Weissman and colleagues in humans that the BCR-ABL gene occurred in a dormant HSC population but was not sufficient to drive blast crisis transformation.^3,8 Rather, malignant reprograming of granulocyte-macrophage progenitors (GMP) into leukemia stem cells (LSCs) by unregulated beta-catenin and CD47 upregulation fueled blast crisis transformation of CML.⁹ Discovery of the leukemia stem cells (LSCs) in AML by Dick and colleagues demonstrated that there were clonal hierarchies that dictated therapeutic resistance as defining feature of human leukemias.¹⁰ Moreover, the indelible yet seemingly capricious effects of environmental stress and aging on human benign, pre-malignant and malignant HSC biology continue to be an enigma.

Over the course of six decades, functional hematopoietic stem cell research has provided a strong rationale for identifying human-specific cell autonomous and non-cell autonomous drivers of accelerated aging, pre-malignant and malignant hematopoiesis and thereby enable the rapid development and implementation of interception strategies that are predicated on preventing pre-leukemia stem cell (pre-LSC) transformation into leukemia stem cells (LSCs).^2-39

Inflammaging and Clonal Hematopoiesis of Indeterminate Potential

An increasingly thorough understanding of the molecular, phenotypic and functional underpinnings of normal HSC homeostasis has provided a framework for elucidating drivers of pre-malignant hematopoiesis in stressful microenvironments.^12,13

As a result of momentous advances in stem cell whole genome and RNA sequencing, combined with single cell spatial genomics, transcriptomics and proteomics and perhaps more importantly transformative stem cell functional analyses that quantify tissue-specific stem cell responses to different environmental exposures, essential insights can be made into intrinsic and extrinsic drivers of HSC aging and pre-leukemic development.

While host innate and adaptive immune responses evolved to protect stem cells and other cells involved in tissue homeostasis from viral and bacterial pathogens, chronic immune activation is associated with systemic signaling driven by pro-inflammatory cytokines, such as tumor necrosis factor (TNFalpha), interferon (IFN alpha, beta, gamma) and interleukins (IL-1, IL-6), by activated T cells and tissue resident macrophages. Both mouse model studies and humanized model systems show that aging is associated with a decrease in neutrophil respiratory burst; a functional decline in macrophage production of Toll-like receptors as well as chemokine and cytokine production resulting in decreased T cell proliferative potential and reduced NK cell activity that leads to diminished immune surveillance against pre-leukemic cells. However, other aspects of immunity increase with aging as evidenced by increased production of pro-inflammatory cytokines by peripheral blood mononuclear cells from elderly compared with young individuals in response to mitogens in vitro (Fagiolo). Moreover, IL-6 levels have been shown to be higher in centenarians. Indeed, chronic inflammation has long been linked with accelerated tissue aging, particularly in the hematopoietic system, and is now termed, inflammaging. However, the role of stem cell inflammaging in HSC homeostasis and pre-leukemic development has not been clearly elucidated.¹³

Recently, both macroenvironmental and microenvironmental drivers of inflammaging in hematopoietic and other tissue-specific stem cells have come to the fore as major arbiters of pre-cancer stem cell generation and evolution to self-renewing cancer stem cells, which evade host innate and adaptive immune responses. Seminal mouse and zebrafish model studies have demonstrated that HSC aging is typified by myeloid lineage bias, reduced dormancy and diminished regenerative (self-renewal) potential. Subsequent studies confirmed that the same is the case for human HSCs and spawned the field of clonal hematopoiesis (CH). While clonal somatic DNA mutations in epigenetic modifier genes, including TET2 and DNMT3A, in stem cell populations increase the risk of developing acute myeloid leukemia (AML) as well as cardiovascular death, there may be some protective effects with regard to Alzheimer’s disease.³ The complexity of clonal stem cell dominance has become apparent as a result of high resolution single cell sequencing that demonstrates that some mutations in splicing factor related genes emerge later in life and provide a greater clonal competitive advantage and potential for AML development than classic epigenetic modifier gene mutations.¹⁵ However, the propensity to develop AML varies substantially between individuals, thereby suggesting that host environmental exposures and immune haplotypes may shape the trajectory of these stem cell clone wars.

Pre-Leukemic Evolution of Hematopoietic Stem Cells

Recent provocative data suggest that hematopoietic stem cell aging may be accelerated by acquisition of somatic DNA mutations early in life and that the usually indolent process of clonal hematopoiesis of indeterminate potential (CHIP) may be superseded later in life by more rapidly dividing, splicing factor gene mutated clones that form the apex of an oligoclonal and ultimately malignant hierarchy.^14-20 While there is substantial variation in the occurrence and clonal trajectories of CHIP between monozygotic twins thereby underscoring the importance of environmental exposures and epigenetic factors in the evolution of CHIP, those with CHIP have shorter telomeres.¹⁵

In addition to radiation and toxic exposure-induced somatic DNA mutagenesis, pre-leukemia stem cells (pre-LSC) generation from hematopoietic stem and progenitor cells may be driven by activation of primate-specific cytidine-to-thymidine (C-to-T) DNA editing enzymes.²¹ As human longevity is extended by advances in precision medicine; the global spread of viral and bacterial pathogens induces acute and chronic inflammatory responses; and immune dysfunction is elicited by advanced age and stem cell stress-inducing environments, including low earth orbit (LEO) as space exploration expands, extrinsic exposures will start to gain even greater relevance with regard to accelerated human aging and pre-leukemia stem cell generation coupled with immune dysfunction.²² These mutations result in cell differentiation (lineage) bias and evasion of apoptosis as well as evasion of host innate and adaptive immune responses (Fig. 3). Recent research performed by our group and others suggests that inflammatory cytokine-activated APOBEC3C induces C-to-T somatic DNA mutagenesis thereby fueling pre-leukemic stem cell generation in myeloproliferative neoplasms (Fig. 4).²¹ This niche-dependent inflammatory cytokine milieu combined with evasion of host innate as well as adaptive immune responses enables pre-leukemic clonal escape and expansion.

Malignant Reprogramming of Progenitors into Leukemia Stem Cells

Cumulative data suggest that some clonal HSC mutations enhance sensitivity to inflammatory growth factor signaling, including JAK2 V617F, MPL and CALR. For the most part, these mutations lead to the generation of myeloproliferative neoplasms (MPNs), which are initiated by pre-leukemic stem cells with myeloid skewed differentiation potential, loss of dormancy and a propensity to migrate to extramedullary niches, including the spleen, thereby resulting in myeloproliferative neoplasm development (Fig. 3, 4). Comparative whole genome sequencing of purified hematopoietic stem cells and mature cells in saliva from the same individuals with myeloproliferative neoplasms suggest that germline mutations may predispose individuals to chronic inflammatory cytokine signaling that enhances inflammaging, CH and pre-leukemic stem cell generation.²¹ Protracted activation of primate-specific anti-viral DNA editing enzymes, such as APOBEC3 cytidine deaminase enzymes, in response to chronic pro-inflammatory cytokine signaling can induce cytidine to thymidine (C-to-T) mutations thereby promoting clonal somatic mutagenesis in stem cell populations.²¹ Moreover, APOBEC3C fueled expansion of the progenitor pool triggers ADAR1 activation resulting in increased RNA editing, including of STAT3, and evasion of tumor suppression. Overexpression of ADAR1 and missplicing of GSK3beta fuel activation of beta-catenin thereby resulting in malignant reprogramming of myeloid progenitors into self-renewing leukemia stem cells that drive blast crisis transformation in chronic myeloid leukemia and myeloproliferative neoplasms (Fig. 5).

Reversal of Malignant Progenitor Reprogramming

Increased expression of the inflammatory cytokine inducible splice isoform of ADAR1, ADAR1 p150 (ADAR-202) has been linked to progression and therapeutic resistance of 20 different malignancies. Moreover, RNA splicing deregulation also promotes leukemia stem cell in both adult and pediatric patients. While splicing modulators show some signs of clinical efficacy, including E7107 and H3B-8800, ocular toxicity or insufficient efficacy against LSC in AML, respectively, have limited their use. Recently, we completed CIRM and NCI-funded pre-IND studies with a selective splicing modulator, Rebecsinib (17S-FD0895), that prevents splicing-mediated activation of ADAR1 into pro-malignant isoform, p150 (Fig. 6).²⁹

Other strategies for inhibiting LSC self-renewal are under development and include N⁶-methyl adenosine RNA targeted therapeutics, small molecule APOBEC3 inhibitors, anti-sense oligonucleotide (ASO) targeting of upstream ADAR1 activators and cytokine signaling disruption with Cirmtuzumab and other LSC-targeted monoclonal antibody therapies.³⁴ Deregulation of sonic hedgehog signaling has also proven to be an LSC Achilles heel with glasdegib targeted small molecule inhibition resulting in doubling of survival for elderly patients with AML.^20,33 Also, deregulated programmed cell removal remains a key arbiter of LSC escape from host innate immune responses. Targeted inhibitors of the “don’t eat me signal”, including CD47 and its ligand, SIRP alpha, are currently undergoing clinical development for LSC eradication in AML. Additional strategies to enhance innate and adaptive immune eradication of LSC include inhibition of immune editing that leads to HLA class 1 loss and induced pluripotent stem cell as well as lipid nanoparticle derived NK cell activation and macrophage repolarization strategies.

In summary, human hematopoietic stem cell informed therapeutic development aimed at inhibiting pre-LSC and LSC propagation may reduce rates of therapeutic resistance and ultimately allow patients to emerge from the cave of uncertainty and see the light of treatment free remission.

References

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