TARGETING LEUKEMIA STEM CELL RESISTANCE IN CHRONIC MYELOGENOUS LEUKEMIA

Abstract

A major limitation of current leukemia treatment is that most patients ultimately relapse. Leukemia cells show heterogeneous potential and response to treatment. We have shown that primitive leukemia stem cells (LSC) in chronic myelogenous leukemia resist elimination by treatment, and persist as a source of relapse. The bone marrow microenvironment (BMM) plays a critical role in of hematopoietic stem cell maintenance and regulation. There is increasing interest in the role of the BMM in promoting LSC maintenance, resistance to therapy, and ultimately disease relapse. Recent studies have shown that leukemia-induced changes in the BMM provide a competitive growth advantage to LSC, and support their preservation after treatment. We are studying mechanisms of niche regulation of LSC to guide development of novel approaches to target LSC and enhance cures.

INTRODUCTION

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy caused by the oncogenic BCR-ABL tyrosine kinase (1). Tyrosine kinase inhibitors (TKIs) are remarkably effective in the treatment of CML and result in remission induction and prolongation of survival in most patients (2). However, TKI treatment does not eliminate the leukemia stem cells (LSC) responsible for leukemia generation (3). As a result, leukemia usually recurs upon drug discontinuation, and most patients require continued treatment to prevent recurrence. Our research is focused on understanding mechanisms of LSC resistance and developing strategies to increase the proportion of CML patients achieving treatment-free remissions.

TARGETED THERAPY FOR CML

CML results from a reciprocal translocation between chromosomes 9 and 22, t(9; 22), resulting in the fusion between the 5' end of the BCR gene and the 3' end of the ABL1 gene leading to the formation of the BCR-ABL oncogene (1,4). The BCR-ABL protein is expressed in CML hematopoietic cells and has constitutive tyrosine kinase activity (4). CML presents in an initial chronic phase characterized by myeloproliferation with retention of differentiation, and progresses to an accelerated phase and a terminal and fatal acute leukemia-like blast crisis phase (1). The development of BCR-ABL TKIs have revolutionized therapy for CML. TKIs have proven highly effective in inducing remission, preventing disease progression, and prolonging survival of CML patients (2).

LEUKEMIA STEM CELLS

Most leukemia patients achieving remission are not cured and ultimately relapse. Relapse is the major contributor to leukemia-related mortality and morbidity, and its human, societal, and financial costs. Genetic and functional heterogeneity of leukemia cells is a major contributor to relapse in leukemia patients. HSCs are multipotent cells can self-renew as well as differentiate into all the different mature blood cell types. Normal hematopoiesis is tightly regulated to maintain steady-state levels of the blood cells through the life of an individual. One aspect of heterogeneity within the broader leukemia cell population is the presence of small populations of LSCs capable of leukemia propagation (5,6). LSCs share several properties with normal HSCs and are resistant to elimination by conventional and targeted therapies. LSCs possess several qualities that are similar to HSCs, including the abilities to self-renew and differentiate. CML has long been recognized to be of stem cell origin (7). Although CML LSCs show enhanced proliferation compared to their normal counterparts, a highly quiescent subpopulation of LSCs has been shown to be present in bone marrow of CML patients (8).

LSC RESISTANCE TO TKIs

TKIs have a strong antiproliferative effect on LSCs, but induce only modest levels of apoptosis (9,10). Quiescent LSCs are especially resistant to TKI-induced apoptosis and elimination. Several studies have found that TKIs effectively inhibit kinase activity within LSCs, indicating that LSC resistance is BCR-ABL kinase-independent (11,12). We and others have shown that BCR-ABL+ stem cells persist in patients in remission on TKI treatment (3). As a result, most CML patients show rapid recurrence of leukemia if TKI treatment is interrupted. On the other hand, some CML patients who achieve and sustain undetectable minimal residual disease (UMRD) with imatinib or other TKI therapy are able to successfully discontinue therapy without leukemia recurrence (13). In this patient population, molecular recurrence-free survival after imatinib discontinuation was 43% at 6 months and 38% at 60 months. However, only a small proportion of CML patients can maintain a treatment-free remission after discontinuing TKI treatment, and most patients require prolonged, likely life-long TKI treatment (5). LSC persistence may also allow for development of acquired TKI resistance, disease relapse, and progression. The risks of prolonged TKI treatment include noncompliance, associated with increased risk of treatment failure and progression; development of treatment intolerance, affecting quality of life, often requiring dose reduction or change in TKI; development of serious vascular complications with second-generation TKIs; and teratogenic effects of TKI, which preclude pregnancy while on treatment (14). Another important consideration is the high cost of TKI treatment which is compounded by the increasing prevalence of CML resulting from improved survival with TKI treatment, and the costs associated with continued, regular molecular monitoring of these patients. In addition, copayment requirements could adversely impact individual patients and affect adherence to treatment (15).

There is a pressing need to better understand mechanisms that allow LSC maintenance and retention of capacity to regenerate leukemia following antileukemia therapy to reduce relapse and enhance cures. Diverse intracellular regulatory mechanisms contributing to CML LSC maintenance and drug resistance have been identified. Mechanisms contributing to TKI resistance in CML LSCs include altered signaling through the JAK/STAT, nuclear factor kappa light chain enhancer of activated B cells (NF-kβ), transforming growth factor β (TGFβ), and β-catenin pathways, transcriptional regulatory networks involving MYC, SIRT1, BCL6, and P53, and epigenetic modulation by PRC2 complexes (5,14). There is evidence that signals from the bone marrow microenvironment (BMM) may also protect LSCs from TKI treatment and regulate their capacity to regenerate leukemia.

MICROENVIRONMENTAL MECHANISMS OF LSC RESISTANCE

HSC homeostasis is regulated by both cell intrinsic mechanisms and extrinsic signals from the BMM. Schofield proposed the concept of the stem cell niche, describing a specialized microenvironment that regulates stem cell function in vivo (16). The nature of the HSC niche within the BMM has been the subject of much investigation, and several cell types, including sinusoidal and arteriolar vascular endothelial cells, subendothelial cells, osteoblastic cells, nonmyelinated Schwann cells, adipocytes, and megakaryocytes have been identified as contributing to HSC regulation (17).

LSCs are also regulated by the cellular and molecular components of the BMM, which can protect LSC from conventional and targeted therapy (18). Genetic lesions in leukemia cells, in addition to providing cell-autonomous growth signals, also alter how LSCs interact with and respond to the BMM. CML LSCs show reduced adhesion to stroma, which may contribute to their abnormal trafficking (19). It is increasingly recognized that leukemic cells can induce changes in the BMM to enhance support of leukemic hematopoiesis at the expense of normal hematopoiesis (20). It is also believed that LSCs may hijack HSC niches to selectively support their own growth. Indeed, BMM alterations can even induce the development of myeloid leukemias by themselves by inducing hematopoietic dysfunction (21). A clear understanding of the role of BMM in the pathogenesis of leukemia is of considerable clinical significance, since understanding BMM mechanisms responsible for LSC maintenance could provide alternative strategies to improve their elimination and enhance survival of patients.

ROLE OF INFLAMMATORY CYTOKINES IN LSC RESISTANCE

We observed increased levels of several inflammatory cytokines in CML bone marrow, including the pro-inflammatory cytokines interleukin (IL)-1α, IL-1b, and tumor necrosis factor α (TNFα) (20). These abnormalities were not fully corrected during TKI-induced remission. Our preliminary studies indicate that inflammation-induced alterations can differentially affect CML and normal stem cell maintenance and growth and provide a competitive advantage to LSCs. We have shown that CML LSCs show increased expression of the IL-1 receptors, IL-1RAP, and IL-1R1, and enhanced IL-1–induced NF-kB activation compared to normal HSCs (22). Inhibition of IL-1 signaling using recombinant IL-1 receptor antagonist (IL-1RA) reduces growth of CML LSCs. The combination of IL-1RA with TKIs results in significantly greater inhibition of CML LSC compared with TKIs alone. These results show an important role for IL-1 signaling in CML LSC maintenance following TKI treatment. We further observed that TNFα also supports survival of CML progenitors by promoting NF-kB activity. Inhibition of autocrine TNFα signaling using a small-molecule TNFα inhibitor induces apoptosis in CML progenitors. TNFα inhibition combined with nilotinib increases apoptosis compared to TKI alone, and reduces quiescent CML LSCs. These results support a role for TNFα signaling in maintenance and TKI resistance in CML LSCs (23). JAK/STAT activity is enhanced in CML LSCs through signaling via cytokines including IL-6. The combination of the JAK2 inhibitor ruxolitinib with nilotinib increases apoptosis in CML progenitors, and reduces primitive, quiescent CML stem cells, while having minimal effects on normal HSC. These results support JAK2 inhibition as a therapeutic strategy to target CML LSCs (24). Single-cell analysis of samples from patients with CML who are undergoing TKI treatment shows that the LSCs with distinct quiescence and HSC-associated gene expression signatures represent a minority of LSCs at diagnosis but are progressively enriched during TKI therapy (25). These LSCs are progressively enriched for TGF-β, TNF-α/NF-κB, and IL-6/STAT3–associated gene expression, consistent with our experimental findings.

ROLE OF STEM CELL REGULATORY FACTORS IN LSC RESISTANCE

Niche regulatory mechanisms regulating LSC fate have not been well studied. We used an osteoblast (OB) ablation mouse model to study the role of OBs in regulating normal HSCs and CML LSCs. OB ablation results in reduction of primitive HSCs. HSCs from OB-ablated mice showed increased cycling and decreased long-term engraftment and self-renewal capacity. In the CML mouse model, OB ablation results in accelerated leukemia development with reduced survival (25). The notch ligand Jagged-1 is over-expressed on CML OB, and culture with Jagged-1 reduces cell cycling in HSCs and LSCs. These studies support a role for OBs in regulating HSC quiescence and self- renewal, and modulating leukemia development. We have further shown that Wnt signaling protects CML LSCs from TKI treatment (26). CML progenitors show increased expression of the Wnt receptor FZD4 and enhanced response to Wnt, compared to normal CD34+ cells. FZD4 knockdown inhibits CML progenitor growth. Secretion of Wnt ligands requires modification by the Porcupine (PORCN) O-acyl transferase. A PORCN inhibitor, WNT974, efficiently antagonizes Wnt signaling in CML CD34+ cells, and in combination with the TKI inhibits proliferation of CML progenitors, and reduces their growth in NSG mice, compared to TKI alone. Treatment of BCR-ABL mice with TKI plus WNT974 reduces LSC numbers and enhances survival after discontinuation of treatment, compared to TKI alone (27). The hedgehog (Hh) signaling pathway is also activated in CML CD34+ cells. LDE225, a small molecule SMO inhibitor inhibits the Hh pathway in CML CD34+ cells, and reduces CML LSC but normal HSC engraftment in NSG mice. Combined LDE225 and TKI treatment of BCR-ABL mice enhances survival and reduces leukemia development. These results indicate that Hh inhibition, in combination with TKI, targets CML LSCs (28). Taken together these studies show a role for alteration of developmental signaling mechanisms in enhanced support of LSC compared to HSC growth by the BMM.

CONCLUSION

The importance of LSCs relates to their critical role in leukemia development, progression, and recurrence, making them key targets of treatment. As discussed, there are compelling reasons to develop strategies to allow discontinuation of lifelong TKI treatment. With our improving understanding of factors that regulate LSC growth, we are recognizing that it may not be necessary to eliminate all LSCs to prevent leukemia recurrence, and that approaches to making the microenvironment less hospitable for LSC support and growth may promote long-term attrition of LSC, or restrain their ability to regenerate leukemia. Improved understanding of LSC regulation will drive continued progress towards cure of leukemia using targeted approaches.

DISCUSSION

Zeidel, Boston: We've got a lot of exposure in our place to dendritic-based fusion vaccines, which seem to have worked pretty well for acute myelogenous leukemia and perhaps a little less for multiple myeloma. It would seem to me that this cries for this kind of intervention because we don't care if the stem cells are there as long as every time they get down to a certain stage they just get killed. I suspect having met 50 patients or more with acute myelogenous leukemia over a decade on no therapy who should be dead — that they have stem cells but they have no symptoms and they're on no medicine. So, is that a strategy that is being thought of for this illness?

Bhatia, Birmingham: I think there's a lot of interest in developing immune-based strategies. There has been work in the past looking at dendritic cells. I am not aware of anyone who is actively pursuing it right now. But it would make a lot of sense.

Tweardy, Houston: The cytokine profile that you describe is conducive for myeloid-derived suppressor cell development. I was wondering if you could comment on any strategies that potentially can target that sort of cell population that can cause tumor anergy. Of course, the other is immune checkpoint and therapies to sort of target the T cell derived on that side of that tumor anergy equation.

Bhatia, Birmingham: Our understanding of the alterations in the immune system that make the difference between maintaining remission and not preventing recurrence are not fully understood. They're the subject of intense investigation. We are just starting to scratch the surface of this. This is really where we want to go in terms of getting a better understanding of what these mechanisms are and then using them to guide us as to what are the best approaches to use.

Wilson, Durham: You mentioned the cost of the medications going up astronomically — out of proportion to inflation. Has your organization or others addressed that?

Bhatia, Birmingham: That's probably the biggest problem that we have right now. There are patient assistance programs that the companies provide. They tend to provide them for more of the newer drugs because they want those to be used as opposed to Gleevec which is going off patent. But even with Gleevec going off patent, the prices haven't really come down substantially. Then copayment can become a bigger sort of problem. There's a lot of work being done in terms of looking at the burden of costs to patients and the role that has in terms of patient adherence and how that affects outcomes. I think it's going to require a degree of advocacy or some other involvement — probably at the health services level and politically — in order to address that. I don't know how easily it will be able to do it nationally but maybe at the state level. The state may be a place that we can make the case of cost savings by investing in making sure that these patients can get these drugs at an affordable cost because, in the end, we may save a lot more money, with patients otherwise progressing and requiring more intense treatments and loss of productivity in other things.