CXCR4 in Acute and Chronic Leukaemia. Dr Vanessa Manitta

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1 CXCR4 in Acute and Chronic Leukaemia Dr Vanessa Manitta

2 small secreted proteins Chemokines Direct the movement of circulating leukocytes to sites of inflammation mation or injury > 40 known chemokines Two groups: Inflammatory chemokines transiently expressed to attract inflammatory cells to sites of inflammation and infection Homeostatic chemokines Constitutively expressed

3 Chemokines Two main subfamilies with different structure and function CC : First 2 of the 4 cysteine residues are adjacent to each other CXC: If 2 cysteine residues are separated by an intervening amino acid Two additional classes of chemokines: lymphotactin (XC) and fractalkine (CX3C)

4 Chemokine Receptors Family of 7 transmembrane domain, G-protein G coupled cell surface receptors chemokine NH2 S-S receptor 20 chemokine receptors Gα β γ induce directional migration of cells towards a gradient of a chemotactic cytokine (chemotaxis( chemotaxis) Phosphatidylinositol 3-kinase (PI3-kinase) and Ras-to to-mapk pathways are the route which the receptors convey their signals

5 Chemotaxis Interactions between chemokine receptors and their chemokines helps coordinate trafficking and organization of cells within various tissue compartments Chemokines on the luminal surface of vascular endothelium can activate chemokine receptors on rolling blood lymphocytes Activation results in a signalling cascade leading to polymerisation and breakdown of actin Results in the formation of lamellipodia Baggiolini Nature 1998

6 Chemotaxis Stimulation also induces the upregulation and activation of integrins Integrins cause initial arrest and firm adhesion of the leukocytes to the endothelial cells on the vessel wall Followed by transendothelial migration where chemokine gradients direct localization and retention of these cells Process called homing

7 Chemotaxis

8 Function of Chemokines Lymphoid organ development Metastasis T H 1/T H 2 differentiation Inflammation Chemokines Wound healing Cell recruitment Leukocyte trafficking Angiogenesis / angiostasis

9 CXCR4 Chemokine receptor Expressed by haematopoietic and non-haematopoietic cells In 1996 found to have a role in the pathogenesis of HIV co-receptor for entry of T-tropic T (X4) HIV into CD4+ T cells. Binds ligand CXCL12 (Stromal( cell-derived factor-1 1 SDF-1)

10 CXCR4 Important role in haematopoiesis, development, and organisation of the immune system CXCR4 and CXCL12 gene knockout mice exhibit identical lethal phenotype Characterised by deficient myelopoiesis,, B-lymphopoiesisB lymphopoiesis, abnormal neuronal and cardiovascular development (Burger et al BJH 2007) Identical phenotype suggests CXCR4 and CXCL12 have a monogamous relationship

11 CXCL12 Homeostatic chemokine Constitutively secreted by marrow stromal cells Potent chemoattractant for various haematopoietic cells, including HSCs, HPCs,, and B lymphocytes Marrow stromal cells are the principal source of CXCL12 Recent studies have characterised an alternate receptor for CXCL12, termed CXCR7 - mediates cell adhesion and survival signals (Burns et al JEM 2006)

12 Role of CXCR4/CXCL12 CXCR4/CXCL12 chemotaxis is essential for 1) marrow specific homing of circulating HPCs and HSCs 2) Retention/maintenance of HSCs in the marrow microenvironment 3) Normal B-cell B development (Broxmeyer et al JEM 2005) 4) Retaining B-cell B precursors in close contact with stromal cells within the haematopoietic microenvironment CXCR4/CXCL12 chemotaxis also required for homing of circulating cardiac, endothelial, and neural progenitors to peripheral tissues

13 HSC Homing Homing of HSCs is restricted to CXCL12 gradients within the marrow and no other cytokine (Wright et al JEM 2002) CXCR4 dependent - HSCs in CXCR4 knockout mice fail to migrate to the bone marrow during fetal development Regulated by tissue hypoxia in the marrow microenvironment and ischaemic tissues Induces production of HIF-1 (Hypoxia inducible factor-1) Results in selective expression of CXCL12 and CXCR4 in direct proportion to reduced oxygen tension Increases the adhesion, migration and homing of circulating CXCR4+ progenitor cells to hypoxic tissue Ceradini et al (Nat med 2004)

14 HSC Maintenance in Bone Marrow HSC reside in hypoxic areas of the marrow along stromal cells Stromal Niches are scattered throughout the intertrabecular space of the marrow cavity - adjacent to a vascular network termed sinusoids Stromal cells are thought to provide attachment sites, and secrete or contain surface bound growth factors Marrow microenvironment provides HSCs and progenitors niches for growth and differentiation Molecular mechanisms of stem cell-stromal cell interactions are not fully understood VLA4 and VLA5 involved in adhesion

15 HSC Homing and Maintenance

16 B Cell Development CXCR4/CXCL12 expression 1) essential for normal B-cell B development Gene deletion of CXCR4 or CXCL12 in mouse models results in severely reduced B lymphopoiesis,, but normal T lymphopoiesis 2) helps retain B-cell B precursors in close contact with stromal cells

17 B Cell Homing In vitro, CXCL12 is chemotactic for pro- and pre-b B cells CXCR4 deficient mice contain reduced numbers of pro-b B and pre-b B cells in the marrow, abnormally high numbers found in the blood thought to be due to a premature release from the marrow (Ma et al Immun 1999) Tokoyoda et al (Immun 2004) demonstrated homing of pre-pro pro-b cells and plasma cells to marrow niches where stromal cells secrete high levels of CXCL12

18 B cell Homing Other developmental stages of B cells leave the marrow and circulate through the secondary lymphatic tissues B- and T-zone T chemokines (CXCL13, CCL19, CCL21) regulate their homing in secondary lymphoid tissues Plasma cell differentiation associated with a coordinated switch in chemokine sensitivity - increased sensitivity to CXCL12 Allows for plasma cell homing back to the marrow.

19 B cell Homing

20 CXCR4 and Malignancy Tumour cells have increased expression of CXCR4 and CXCL12 Main actions of CXCL12 in tumours: 1) promotes tumour cell growth in a paracrine fashion by directly stimulating tumour cells via CXCR4. 2) induces recruitment of endothelial progenitors - allows for tumour angiogenesis (endocrine effect of CXCL12) 3) CXCL12 gradients attract CXCR4+ tumour cells to marrow niches High levels of CXCL12 are expressed within hypoxic areas of tumours urs by carcinoma-associated associated fibroblasts (CAFs( CAFs)

21 CXCR4 and Malignancy CXCR4+ tumour cells competes with HPCs for homing and retention within the marrow Tumour cells can displace HPCs/HSCs from their protective microenvironment, resulting in hematopoietic dysfunction. Tumour cells can migrate beneath stromal cells in the marrow and interact with various extracellular matrix adhesion molecules Homing of tumour and leukemia cells provides them with conditions that favour their growth and survival; Protection from spontaneous and chemotherapy induced apoptotic signals

22 CXCR4/CXCL12 and Malignancy

23 CXCR4 In ALL CXCR4 is functional in B cell ALL induces leukaemia cell chemotaxis to CXCL12 Induces spontaneous migration beneath CXCL12-secreting stromal cells Migration requires leukaemia cell expression of CXCR4 and adhesion molecules - VLA4 integrin type (Burger et al et al Blood 1999) Migratory response of ALL cells to CXCL12 depends on p38 mitogen-activated protein kinase signalling (Bendall et al Canc Canc res 2005) Sipkins et al (Nature 2005) direct in vivo evidence that CXCR4 is necessary for the homing of ALL cells to the marrow microenvironment

24 CXCR4 in AML AML cells express functional CXCR4 Expression relatively lower in AML than ALL Differential expression in AML lower expression in M0 (undifferentiated), M1/2 (myeloid), M6 (erythroid) higher expression in M4/5 (myelomonocytic( myelomonocytic) ) and M3 (promyelocytic) (Mohle et al BJH 2000) CXCR4-CXCL12 CXCL12 axis plays a significant role in trafficking and tissue dissemination of AML cells in vivo

25 CXCR4 in AML Burger et al (BJH 2003) CXCR4 co-operates operates with VLA-4 4 during initial AML cell adhesion CXCR4 induces leukemia cell chemotaxis and migration beneath marrow stromal cells Migration of AML cells beneath marrow stromal cells was associated with cell cycle arrest and reduced numbers of cell divisions? Potential mechanism of leukaemia cell evasion from chemotherapy Polymorphism in CXCL12 coding gene associated with higher counts of circulating AML cells and frequency of extramedullary disease (Burger et al BJH 2007)

26 CXCR4 in AML In vitro studies by Zeng et al (Mol Can therap 2006) demonstrated that upon adhesion to stromal cells, AML cells become resistant to spontaneous or drug-induced apoptosis? Mechanism to explain minimal residual disease and subsequent relapses

27 CXCR4 in CLL CXCR4 is overexpressed and functionally active in B-CLL B cells allows for their recirculation between the blood, marrow and lymphoid tissues Burger et al (Blood 1999) - analysed the migratory patterns of B-CLL B CXCL12/CXCR4 interaction induced CLL cell migration beneath stromal cells in CXCR4 dependent fashion Chemotaxis to CXCL12 could be inhibited by monoclonal antibodies against CXCR4 Conclusion: CXCR4 may contribute to the bone marrow tropism of B-CLL B cells and heterotypic adherence to marrow stromal cells

28 CXCR4 in CLL Burger et al (Blood 2000) analysed regulatory factors involved the survival of CLL cells Subset of blood cells from patients with B-CLL B spontaneously differentiates to stromal cells (NLC) which express CXCL12 Upon adhesion to stromal cells B-CLL B cells downmodulate their CXCR4 receptors Adhesion protects leukemic cells from spontaneous apoptosis Survival of B-CLL B cells rapidly deteriorated when separated from their stromal cells effect mitigated by addition of exogenous CXCL12 Conclusions : In vitro, stromal cells directly stimulates CLL cell survival Likely similar role in vivo Increased survival is a CXCL12 CXCR4 dependent process

29 CXCR4 in CLL Burger et al (Blood 2005) - analysed the effect of CXCR4 antagonists on B-CLL B cells stromal cells protected CLL cells from chemotherapy induced apoptosis CXCR4 antagonists resensitised CLL cells cultured with stromal cells to fludarabine-induced induced apoptosis Conclusions CXCL12/CXCR4 not only functions as a chemoattractant but also a survival factor for CLL cells Stromal cells provide a marrow microenvironment that may protect CLL cells from spontaneous and drug-induced apoptosis.

30 CXCR4 in Myeloma B cell differentiation into plasma cells leads to a change in their chemokine responsivenes More sensitive to CXCL12; Lose responsiveness to B-B and T-zone T chemokines MM cells display functional CXCR4 that cooperate with VLA-4 integrins in cell adhesion and migration Gene deleted mice lacking CXCR4 in B cells still have substantial numbers of long lived plasma cells found in the marrow? due to different chemokine requirements for different plasma cell populations generated during immune response Animal models suggest the CXCR4 is not a stringent requirement for f plasma cell homing to the marrow

31 CXCR4 in lymphoma CXCR4 expression demonstrated in B-cell B and T-cell T NHL B-cell NHL also express CXCR3, CXCR5, CCR7, CCR5 CXCL12 enhances migration of follicular NHL cells but not normal germinal centre B cells Distinct pattern of chemokine receptor expression thought to be involved in lymphoma cell trafficking and homing may allow to distinguish different NHL subsets

32 CXCR4 in CML Transfection of BCR-ABL fusion protein into different haematopoietic cells inhibited chemotaxis and CXCR4 pathway Resulted in disrupted signalling and chemotaxis in response to CXCL12 Geay et al (Cans res 2005), Salgia et al (Blood 1999) This disrupted signalling can be restored by inhibition of BCR/ABL BL tyrosine kinase with imatinib (Ptsaznik et al JEM 2002) Conclusions Leukemic Ph+CD34+ cells in CML patients display reduced chemotaxis to CXCL12 when compared to normal CD34+ cells, due to deregulated CXCR4 signalling caused by cross talk between BCR/ABL oncoprotein and CXCR4 May explain the release of CML stem cells out of the bone marrow and into extramedullary sites

33 CXCR4 in nonhaematopoietic malignancies In vitro studies of breast cancer cells CXCL12 highly expressed by stromal fibroblasts within lymph nodes, lung, liver and marrow (primary sites for metastatic spread) express higher levels of functional CXCR4 receptors than normal breast tissue associated with relatively poorer overall survival in patients In vitro studies of SCLC cells CXCR4 is the major chemokine receptor CXCR4 activation induced migratory and invasive responses, and adhesion to marrow stromal cells in a CXCR4 and integrin dependent fashion. Adhesion protected SCLC cells from chemotherapy induced apoptosis.

34 Prognostic implications of CXCR4 CXCR4 expression levels on leukemic cells can be determined by flow cytometry. Anti-CXCR4 antibody 12G5 is the most widely utilised antibody clone to detect CXCR4 Studies have investigated correlations between CXCR4 expression and outcome and/or specific clinical presentations in leukaemia patients

35 Prognostic implications of CXCR4 In ALL Crazzolara et al (BJH 2001) Retrospective study on childhood ALL CXCR4 levels on ALL cells correlated with extramedullary organ infiltration Independent predictor for extramedullary disease from the peripheral lymphoblast count. Alternatively Schneider et al (BJH 2002) detected a correlation between CXCR4 expression and WBC count and organ infiltration at diagnosis no correlation between CXCR4 expression and extramedullary relapses? Discrepancy between studies due to small patient numbers and different treatment protocols Relevance of CXCR4 in ALL needs to be re-evaluated evaluated in larger trials

36 Prognostic implications of CXCR4 In AML Rombouts et al (Blood 2004) Retrospective study assessing BM samples of 90 adult AML patients CXCL12/CXCR4 interaction required for the survival of myeloid differentiating cells Co-expression of CXCR4 and CD34 had a significantly reduced survival and higher probability of relapse CXCR4 expression was significantly higher in Flt3/ITD AML than in i Flt3/wildtype AML Multivariate analysis: predictive value of CXCR4 expression was independent of prognostic markers such as age and cytogenetic abnormalities FLT3/ITD was not independent from CXCR4 for predicting RFS? FLT3/ITD could be a subordinate of CXCR4 expression

37 Prognostic implications of CXCR4 In AML Prospective study by Spoo et al (Blood 2007) evaluated the prognostic implication of CXCR4 in 90 pts with AML lower CXCR4 expression had a significantly longer RFS and OS than patients who displayed intermediate or high CXCR4 levels OS /- 2.9 months for low CXCR4, /- 2 months for high expression Multivariate analysis: CXCR4 is a prognostic marker that is independent of other previously established prognostic markers (eg( cytogenetics abnormalities, LDH, leukocytosis or age).

38 Prognostic implications of CXCR4 In AML

39 Prognostic implications of CXCR4 Konoplev et al (Cancer 2007) Retrospective study In AML 122 AML patients with normal karyotype and unmutated FLT3 gene to determine prognostic impact of CXCR4 independent of FLT3 CXCR4 expression, presence of multilineage dysplasia and high creatinine level predicted poorer overall and event free survival

40 Prognostic implications of CXCR4 Conclusions : In AML CXCR4 expression on AML cells is an adverse prognostic indicator in AML that is independent from other prognostic factors

41 Prognostic implications of CXCR4 In CLL CXCR4 expression levels reported to correlate with WBC counts, numbers of circulating CLL cells and disease stage (Burger et al BJH 2007) In 39 pts with familial CLL, CXCR4 levels had a negative prognostic impact (Ishibe et al Blood 2002) Clinical trials required to clarify the importance of CXCR4 as a predictor for disease stage and prognosis

42 CXCR4 antagonists Initially administered to human subjects in an attempt to develop p a novel treatment of HIV No benefit regarding the viral load investigation ceased. Investigators noticed elevated WBC counts and mobilisation of CD34+ HPC into the bloodstream Two agents in clinical trials AMD3100; ALX40-4C 4C T140 : 14 residue polypeptide

43 CXCR4 antagonists Compounds that target the CXCR4 receptor or its ligand could 1) disrupt interactions between leukaemia cells and their protective tive stromal counterparts AML patients who underwent autologous stem cell mobilisation with AMD leukemia cells mobilised into the circulation (Andreeff et al Blood 2006) 2) Strongly inhibit migratory and signalling responses to CXCL12 CXCR4 antagonists inhibited the homing of leukaemia cells to the marrow in animal models (Sipkins et al Nature 2005) 3) antagonise paracrine growth and survival effects of CXCL12 4) make leukaemia cells more accessible to conventional therapy

44 CXCR4 antagonists in CLL Burger et al (Blood 2005) - evaluated T140 and its analogs for their capacity to inhibit CXCL12 responses in CLL cells. T140 inhibited actin polymerisation, chemotaxis and migration of CLL cells beneath stromal cells. antiapoptotic effect of synthetic CXCL12 antagonised stromal cell mediated protection of CLL cells from spontaneous and chemotherapy induced apoptosis was antagonised Conclusion: CXCR4 antagonists effectively block CXCL12 induced activation, migration and signalling of CLL cells and made them more susceptible to spontaneous and chemotherapy induced apoptosis

45 CXCR4 antagonists in ALL Juarex et al (Leukaemia 2003) examined the capacity of several CXCR4 inhibitors to block CXCL12 driven responses in pre-b B ALL cells in vitro. migration of ALL cells to CXCL12 secreting marrow stromal cells was blocked stromal cell-mediated protection of ALL cells from cytotoxic agents was partially disrupted. proliferation of pre-b B ALL cells on bone marrow stroma was reduced little effect of CXCL12 antagonists on the stromal-dependent survival of the pre-b B ALL samples tested

46 CXCR4 antagonists and HSC mobilisation AMD3100 currently evaluated in phase III clinical trials for mobilisation of HSC AMD3100 currently evaluated in preclinical studies for treatment of neoplastic or autoimmune diseases. Broxmeyer et al (J exp med 2005) AMD3100 could mobilize HPCS/HSCs HSCs rapidly from human and murine systems synergised with G-CSF G to enhance mobilisation

47 CXCR4 antagonists and HSC mobilisation Liles et al (Blood2003) : AMD3100 in 26 healthy volunteers induced a rapid, generalized leukocytosis associated with a transient (up to 10 fold) increase in peripheral blood CD34+ cells ls Dose dependent Devine et al (JCO 2004) reported AMD3100 safe and effective agent for the mobilization of CD34+ cells in patients with MM or lymphoma who have received prior chemotherapy

48 CXCR4 antagonists Agents well tolerated SE headache, perioral paresthesia, nausea; all resolved in 24hours Concern with co-administration with cytotoxic drugs normal haematopoietic progenitors that are normally protected in the marrow microenvironment would be exposed to the toxicity of chemotherapy? A leukaemia cell targeted therapy eg. anticd20 or anticd52 monoclonal antibody in combination with CXCR4 antagonist could avoid this hazard.

49 Summary CXCR4 is essential for marrow specific homing and maintenance of circulating HSCs CXCR4 is essential for normal B-cell B development and retaining/maintaining B-cell B precursors and plasma cells within the haematopoietic microenvironment Tumour cells can hijack the CXCR4/CXCL12 chemotaxis mechanism Allows for homing of tumour cells to the marrow microenvironment providing them with conditions that favour their growth and survival provides anti-apoptotic apoptotic signals and confer drug resistance? Mechanism to explain minimal residual disease and subsequent relapses

50 Summary Studies establish CXCR4 expression as an independent prognostic marker in AML Should CXCR4 expression be incorporated into the initial diagnostic workup of AML pts on clinical trials? Further trials required in ALL and CLL in determining prognostic significance of CXCR4 CXCR4 antagonists promising -inhibiting the adhesion and migration of tumour cells into the marrow protective microenvironment CXCR4 antagonists promising alternative or adjuvant HSC mobilisation