by Chaya Venkat
Most of us depend on the monthly CBC to give us some idea of the status of our CLL. Sometimes, that can be very misleading. CLL is a very well distributed disease, in the bone marrow, lymph nodes, spleen, liver and blood. If you happen to have a variety of CLL that likes to live in the bone marrow and lymph nodes, the WBC numbers you see in your monthly blood test are going to be misleading, in the sense that there are a lot more CLL than you would expect, just from the CBC numbers.
It has become now become clear that while it is relatively easy to kill CLL cells swimming around in the blood, clearing them out of bone marrow and lymphatic system is a lot harder. If you are one of the patients we have been discussing in the last two articles, with deletion of 11q or 17p as the chromosomal aberration driving your CLL, two of the most promising therapy options in your arsenal are the monoclonals, Rituxan and Campath. Both do a very good job of clearing out CLL from peripheral blood, and while Campath does a better job of clearing out the bone marrow than Rituxan, neither of the two monoclonals has an easy time clearing out lymph nodes.
No Cell Is an Island Unto Itself
Why are CLL cells harder to kill in the bone marrow and lymph nodes? Cells of your body are social creatures, living in the midst of their neighbors and constantly exchanging messages and signals with each other. All aspects of their lives, whether to rest, grow, procreate, live or die, pull up stakes migrate to another part of the body, all of these choices are made in the context of signaling between the individual cell and those surrounding it. CLL cells also communicate with their neighbors, except that in their case the signaling has gone haywire, they seem to have tuned out all signals coming in telling them to die. This selective deafness to apoptosis (suicide) signals is at the very heart of their survival advantage. Ominously, they have learned one more trick. Like some ego-driven CEO, they have learned to surround themselves with sycophantic "nurse-like cells" that nurture them and feed their egos, none of the nasty "why don't you do every one a favor and kill yourself" stuff. These nurse like cells are present in the bone marrow and lymph nodes. The protective environment of these locations allows CLL cells to thrive and proliferate, and resist drugs trying to kill them.It should come as no surprise to anyone that the particularly aggressive variants of CLL, such as the ones with 11q deletion and 17p deletion, are also the ones that are more prone to be located in the bone marrow and lymphatic system.
Blood. 2000 Oct 15;96(8):2655-63.
Blood-derived nurse-like cells protect chronic lymphocytic leukemia B cells from spontaneous apoptosis through stromal cell-derived factor-1.
Burger JA, Tsukada N, Burger M, Zvaifler NJ, Dell'Aquila M, Kipps TJ.
Department of Medicine, Division of Hematology/Oncology, University of California at San Diego, La Jolla, 92093-0663.
A subset of blood cells from patients with B-cell chronic lymphocytic leukemia (CLL) spontaneously differentiates in vitro into large, round, or fibroblast-like adherent cells that display stromal cell markers, namely vimentin and STRO-1. These cells also express stromal cell-derived factor-1 (SDF-1), a CXC chemokine that ordinarily is secreted by marrow stromal cells. Leukemia B cells attach to these blood-derived adherent cells, down-modulate their receptors for SDF-1 (CXCR4), and are protected from undergoing spontaneous apoptosis in vitro. Neutralizing antibodies to SDF-1 inhibit this effect. Moreover, the rapid deterioration in the survival of CLL B cells, when separated from such cells, is mitigated by exogenous SDF-1. This chemokine also results in the rapid down-modulation of CXCR4 and activation of p44/42 mitogen-activated protein-kinase (ERK 1/2) by CLL B cells in vitro. It is concluded that the blood of patients with CLL contains cells that can differentiate into adherent nurse-like cells that protect leukemia cells from undergoing spontaneous apoptosis through an SDF-1-dependent mechanism. In addition to its recently recognized role in CLL B-cell migration, SDF-1-mediated CLL B-cell activation has to be considered a new mechanism involved in the microenvironmental regulation of CLL B-cell survival. (Blood. 2000;96:2655-2663)
Following the Trail of Bread Crumbs Home
How do cells know where to go? Think of a CLL cell in your peripheral blood, moving along with the rest of the blood in one of your capillaries. How does it know when it is getting near a lymph node, that it is time to get off the bus? Welcome to the fascinating world of chemokines, adhesion factors and chemical homing signals. This is a rapidly growing field in just the last few years and already the complexity is mind bending. The long-winded pedantic teacher in me cringes at this, but I am going to get right down to the main course, forgo the technical explanations of how lymphocytes go home.
Semin Immunol. 2002 Apr;14(2):83-92.
Chemokines in rapid leukocyte adhesion triggering and migration.
Johnston B, Butcher EC.
Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5324
Leukocyte subsets are recruited from the blood to lymphoid and non-lymphoid tissues via a multi-step process that involves distinct adhesive and activation steps. Chemokines, a family of chemotactic cytokines that signal through G-protein-coupled receptors, play critical roles in regulating the leukocyte recruitment cascade. Chemokines can be transported and immobilized on the surface of vascular endothelial cells, where they activate leukocyte subsets expressing specific receptors. Activation signals induce firm adhesion of rolling leukocytes by rapidly upregulating integrin affinity and/or avidity. Chemokines can also direct migration of adherent cells across the endothelium, and control segregation of cells into specific microenvironments within tissues. The regulated expression of chemokines and their receptors is a critical determinant for homing of specialized lymphocyte subsets, and controls both tissue and inflammation-specific immune processes. Copyright 2002 Elsevier Science Ltd. All rights reserved.
It turns out that one of the most important "homing" mechanisms is a molecule called SDF-1 (Stromal cell Derived Factor, and just to make it more complicated, more recent papers use the acronym CXCR12 for the same molecule). SDF-1 is present in bone marrow, lymph nodes, many epithelial cell walls and tissues. The one-and-only monogamous receptor for SDF-1 is called CXCR4. Any cell carrying the CXCR4 receptor in the neighborhood of SDF-1 finds itself irresistibly drawn to its siren call. In addition to B-cells, CXCR-4 receptor is carried by T-cells, neutrophils and stem cells etc. All of these cells wander around in the peripheral blood for a while, then are called back home to the bone marrow and lymph nodes, there to rest, grow and multiply. CLL cells have many more CXCR-4 receptors on them than normal cells, so they are that much more likely to scurry off and sit in the bone marrow or lymph nodes.
Leukemia. 1999 Dec;13(12):1954-9.
Overexpression of the chemokine receptor CXCR4 in B cell chronic lymphocytic leukemia is associated with increased functional response to stromal cell-derived factor-1 (SDF-1).
Mohle R, Failenschmid C, Bautz F, Kanz L.
Department of Medicine II, University of Tubingen, Tubingen, Germany.
The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) play an important role in trafficking of normal lymphocytes, monocytes, as well as hematopoietic stem- and progenitor cells. SDF-1 constitutively produced by bone marrow stromal cells acts as a chemoattractant supporting the homing of stem cells and may also contribute to the tropism of malignant cells for the bone marrow. Low-grade lymphoproliferative disorders, particularly B cell chronic lymphocytic leukemia (B-CLL), are characterized by the presence of bone marrow infiltration. Therefore, we analyzed expression of the chemokine receptor CXCR4 in B-CLL, and investigated the functional effect of SDF-1 on the malignant cells. By flow cytometry, CXCR4 was consistently expressed on circulating CLL cells at a fluorescence intensity four-fold greater than that of normal B cells, and three-fold greater than that of CD19+/CD5+ cells from the normal bone marrow. CXCR4 was functionally active as demonstrated by a rapid flux of intracellular free calcium in response to SDF-1, which was significantly reduced by the partially blocking CXCR4 antibody 12G5. Moreover, transendothelial migration of B-CLL cells in vitro was stimulated by conditioned medium from bone marrow stromal cells due to its content of SDF-1, as suggested by reduced migration after addition of the CXCR4 antibody 12G5. In accordance with the CXCR4 overexpression, migration of CLL cells was more efficiently stimulated by recombinant SDF-1 compared to migration of normal B cells. We conclude that CXCR4 is overexpressed and functionally active in B-CLL, and may therefore contribute to the tropism of B-CLL cells for the bone marrow stroma.
Blood. 1999 Dec 1;94(11):3658-67.
Chronic lymphocytic leukemia B cells express functional CXCR4 chemokine receptors that mediate spontaneous migration beneath bone marrow stromal cells.
Burger JA, Burger M, Kipps TJ.
Department of Medicine, the Division of Hematology/Oncology, University of California, San Diego, La Jolla, CA 92093-0663
Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1alpha, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.
Don't Let ET Phone Home
If we can disrupt the SDF-1 and CXCR-4 mating, can we throw a monkey wrench into the works? There is a drug called AMD3100 ( I will tell you later why someone invented this drug), which does just one simple thing: it attaches to CXCR-4 receptor and blocks it. The drug has no killing power, it does nothing more than just cover up the CXCR-4 port on cells. In one fascinating experiment, AMD3100 was given to some healthy volunteers. Nothing happened, no toxicity, no side effects, no cell kill. Just one very important observation, each of the volunteers had their WBC number double or even triple. Before you freak-out, just think for a moment. No new lymphocytes were created suddenly, it is just that those sitting comfortably in the marrow or lymphatic systems of these healthy volunteers suddenly found themselves floating loose, their Velcro connection of SDF-1 and CXCR-4 no longer intact. The peripheral blood WBC went up as these cells got flushed out into the open. In a few days, as the AMD3100 wore off and CXCR-4 was once again able to receive signals, the WBC of the volunteers went back to what it was.
How can we use this in fighting CLL? The only research I have found thus far is a wonderful study using AMD3100 for Non-Hodgkin's Lymphoma. For now, this cousin of CLL will have to do as a substitute for CLL. Here are the highlights of this very interesting paper, the abstract is also given below. I urge you to read the whole paper if possible, it is available free at: http://cancerres.aacrjournals.org/cgi/content/full/62/11/3106.
The authors have this closing observation, we should be doing clinical trials with CXCR4 blockers in NHL patients. Yeah, right on!!
Cancer Res. 2002 Jun 1;62(11):3106-12.
CXCR4 neutralization, a novel therapeutic approach for non-Hodgkin's lymphoma.
Bertolini F, Dell'Agnola C, Mancuso P, Rabascio C, Burlini A, Monestiroli S, Gobbi A, Pruneri G, Martinelli G.
Divisions of Hematology-Oncology, Experimental Oncology-IFOM Institute of Molecular Oncology, European Institute of Oncology, via Ripamonti 435, 20141 Milan, Italy.
The chemokine stromal cell-derived factor-1 (CXCL12/SDF-1) and its monogamous receptor CXCR4 are involved in trafficking of B cells and hematopoietic progenitors. CXCR4 expression was found in the large majority of non-Hodgkin's lymphoma (NHL) cell lines and primary cells, and CXCR4 neutralization by monoclonal antibodies had profound in vitro effects on NHL cells including inhibition of transendothelial/stromal migration, enhanced apoptosis, decreased proliferation, and inhibition of pseudopodia formation. In a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) mouse model of human high-grade NHL, CXCR4 neutralization had an impressive efficacy. In a first tumor-challenge trial, CXCR4 neutralization of Namalwa cells injected i.p. delayed tumor growth and reduced tumor weight. In a second tumor-challenge trial, NOD/SCID mice received Namalwa cells i.v. All of the controls died of neoplasia within day 36, whereas 83% of mice injected with cells incubated with anti-CXCR4 were still alive and disease-free >150 days after transplant. The crucial role of CXCR4 in tumor cell extravasation was confirmed by the finding that CXCR4 neutralization before i.v. injection of Namalwa cells in NOD/SCID mice increased the number of cancer cells circulating 24 h after injection. In additional preclinical trials, the therapeutic effect of anti-CXCR4 antibodies was evaluated in mice bearing Namalwa cells injected 3 days before. Tumor growth was abrogated in the majority of treated mice and significantly delayed in the remaining group. Taken together, these data support clinical studies on CXCR4 neutralization in NHL patients by monoclonal antibodies or CXCR4 antagonists.
Some Futuristic Ideas
Now I would like to get a little goofy here, imagine how we can possible use this technology for CLL patients. I realize I am being terribly simplistic and naive, there are a bunch of details and hurdles to overcome, but hey, what is wrong with kicking around a few ideas?
AMD3100 and later versions of the same class of drugs are heavily researched because of their impact in AIDs. Turns out the AIDs virus likes the CXCR4 port as an entry port into T-cells, and it was thought that blocking this port will make it harder for the virus to get into cells. As with all things having to do with AIDs virus, the drugs work, sort of, but the virus has more tricks up its sleeve. It can mutate to use other ports, and blocking all the port has to be heavy duty, otherwise the virus gets in anyway. In the case of CLL, we have the advantage of a more indolent disease (yeah, CLL is the "good cancer" to have!) so these drugs may work better for us. Remember, the slow progression of early stage CLL means there is just a small mismatch in the rate at which CLL cells are born, and the rate at which they die. If we can nudge that equilibrium even just a little, we can hope to change the face of this monster.
I have not heard of any clinical trials using CXCR4 blockers in CLL, yet. But I am willing to bet the pre-clinical trials are under way, and I will be watching for information on this front. But AMD3100 has been given orphan-drug status for one interesting application related to CLL. When a patient undergoes autologous bone marrow transplants, stem cells are collected from the patient's own blood, stored, and grafted back at a later stage. Often, the problem is that not enough stem cells can be harvested. Here is where AMD3100 comes in. Remember, I told you B-cells were not the only ones that express CXCR4. When you flush out the bone marrow by using CXCR4 blockers, stem cells are flushed out too. This means autologous BMT patients have a better chance of getting a good harvest of stem cells from their own blood. This is no small thing, and now they are also talking about using CXCR4 blockers in stem cell donors for allogeneic BMTs, to make the job of collecting stem cells from the donors that much easier.
Still No Free Lunch
There is truly no free lunch. CXCR4 blockers make it easy for B-cells, T-cells, platelets precursors, neutrophils etc to get flushed out of the bone marrow. What does this mean? Since platelets, neutrophils, red blood cells etc need their time in the bone marrow to grow and reproduce, complete and long lasting blockade of CXCR4 receptors on all of these cell lines will mean reduced production of these necessary cells as well. Since cancer cells fortunately express a lot more CXCR4 receptors than normal cells, partial blockade of this receptor means the cancer cells will get hit much more than the normal cells. It is once again a question of dosage, everything in moderation.
Blood. 1998 Aug 1;92(3):756-64.
The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion.
Wang JF, Liu ZY, Groopman JE.
Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
CXCR4 is the receptor for the alpha-chemokine stromal cell-derived factor 1 (SDF-1) and has been shown to be expressed on a diversity of leukocytes. In this report, the expression of the CXCR4 receptor in cells of megakaryocytic lineage and the role of SDF-1 in megakaryocytopoiesis were investigated. Using flow cytometry in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we observed that bone marrow CD34(+), CD61(+) cells, blood platelets, and megakaryocytic leukemia cell lines all expressed the CXCR4 receptor. To examine the expression of the CXCR4 receptor on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-Meg]), CXCR4-positive and -negative CD34(+) populations were separated from bone marrow and cultured in a plasma clot culture system. A subpopulation of the CFU-Meg was found in the CXCR4-positive fraction. The functional significance of CXCR4 expression on cells of the megakaryocytic lineage was examined by studying the effects of SDF-1alpha on migration and proliferation of megakaryocyte progenitor cells in vitro. We found that SDF-1alpha potently induced megakaryocyte progenitor migration and significantly enhanced adhesion of mature marrow megakaryocytes to endothelium. No marked effects of SDF-1alpha alone or in combination with thrombopoietin and stem cell factor/kit ligand on megakaryocyte production in vitro were noted. These results demonstrate for the first time that the CXCR4 alpha-chemokine receptor is expressed on cells of the megakaryocytic lineage from progenitors to platelets and that its ligand SDF-1alpha may modulate several aspects of megakaryocytopoiesis.
We have discussed other important chemokine and receptor pairs in past articles. One example is the LTD4 and CysLT1 pair that is deeply implicated in chronic inflammations such as asthma (Chronic Inflammation and What You Can Do About It) The VEGF and its receptor VEGFR are important in angiogenesis and tumor growth, (Do you like drinking green tea?) as are COX-2 and its receptor. It is now almost a cliché that the best way of handling cancer is by attacking it from a number of directions all at once. Use just one approach, and like squeezing a tube of toothpaste, the malignancy finds ways around the pressure point. Whether it be the recent trend towards combinations of drugs in chemotherapy, and combinations involving monoclonals and standards chemotherapy drugs, the strategy is still to attack on a number of fronts.
This strategy is likely to be true as well in efforts at chemoprevention. We have discussed the effectiveness of green tea extracts, curcumin from the spice turmeric, resveratrol from grapes, soy isoflavones, CysLT1 blockers such as asthma drugs Singulair, and flavones like quercetin. Many of these have some amount of cytotoxicity towards cancer cells, but their mechanism of action is also determined by their ability to block one or more of the signaling mechanisms of cancer cells. Isolate cancer cells from their neighbors, blind them and make them deaf, unable to interact with their surroundings, and they seem to wither away and die. Apoptosis (cell suicide) mechanisms that are faulty because of mutations in ATM or TP53genes seem to be over-ruled and cell death kicks into action under such torture. Kick up cancer cell apoptosis a notch or two, and if we can also make sure to keep them out of their warm and nurturing home in the bone marrow and lymph nodes so that they have no chance to proliferate and make babies, we might well be on our way to controlling indolent cancers like CLL.
I don't know about you folks, but if I had a few million dollars lying around, I would get this research moving on CXCR4 blockers in treating CLL. We need a CLL version of a Mike Milken or Sam Walton, some one rich with his life riding on ways of dealing with a challenging ATM or p53 deletion and bulky lymph nodes. If you know some one that fits this description, make sure you bring CLL Topics and this article to his / her attention, OK? Maybe, after Project Alpha geared towards early stage and good prognosis patients, we can do Project Omega, targeting late stage and poor prognosis patients, with emphasis on flushing out CLL cells from lymph nodes and bone marrow. No one ever accused me of being too timid when it comes to a good fight....