Targeting CXCR4 with Cell-Penetrating Pepducins Enhances Survival in Disseminated Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4244-4244
Author(s):  
Lidija Covic ◽  
Katie M O’Callaghan ◽  
Nga Ngyen ◽  
Hsieh Mo-Ying ◽  
Andreas K. Klein ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Apoptotic Cell ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Intracellular Loop ◽  
Hematopoietic Stem ◽  
Lymphoid Malignancies ◽  
Cell Penetrating ◽  
New Treatment

Abstract Abstract 4244 The G-protein coupled receptor, CXCR4, which normally regulates interactions between stroma and hematopoietic stem cells in the bone marrow, is highly expressed on a variety of malignant hematological cells, such as lymphoma and lymphocytic leukemia cells. A new treatment concept has arisen that CXCR4 may be an effective therapeutic target as an adjunct to standard clinical treatments of hematologic malignancies. In this study we developed novel cell-penetrating lipopeptide antagonists of CXCR4, called pepducins, to interdict signaling to intracellular G-proteins in response to the CXCR4 ligand, SDF-1. We demonstrated that pepducins targeting the first (i1) or third (i3) intracellular loop of CXCR4 completely abrogated SDF-1-mediated chemotaxis of lymphocytic leukemia and lymphoma cell lines, as well as primary cells isolated from patients with chronic lymphocytic leukemia. Pepducins enhanced apoptotic cell death in lymphoma and primary leukemia cells when used in combination with the CD20-targeted antibody, rituximab. Furthermore, pepducin treatment significantly increased survival both as monotherapy and in combination with rituximab in mice with disseminated lymphoma. Together, these data demonstrate that CXCR4/SDF-1 signaling is effectively inhibited by cell-penetrating pepducins, suggesting that these lipopeptide antagonists may represent a potentially new treatment strategy for lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

C-Myc Is Required for Acute but Not for Chronic Hematopoietic Malignances in Pten-Null Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1632-1632
Author(s):  
Yinshi Guo ◽  
Christopher Seet ◽  
Chao Niu ◽  
Peter Breslin ◽  
Shubin Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Conflicts Of Interest ◽  
Apoptotic Cell ◽  
Akt Signaling ◽  
Apoptotic Cell Death ◽  
Lymphocytic Leukemia ◽  
Mutant Mice ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem

Abstract Abstract 1632 Poster Board I-658 c-Myc, the product of an oncogene, is a common target of most leukemic oncoproteins. Deregulation of c-Myc is commonly found in human leukemic blasts. Transgenic over-expression of c-Myc induces myeloid, erythroid, and lymphocytic leukemia in mice, however whether c-Myc is absolutely required for leukemogenesis has not yet been addressed. Pten, a tumor-suppressing phosphatase, inhibits cell proliferation and promotes apoptotic cell death through repression of PI3K-Akt signaling. Inactivating mutations of Pten and deregulation of PI3K/Akt signaling are both involved in the development of both chronic and acute hematopoietic malignances. Mice with Pten deletions in hematopoietic stem cells (HSCs) develop myeloproliferative disorders (MPD) followed by acute T lymphocytic or myeloid leukemia, reminiscent of disease progression in human chronic myelogenous leukemia. Mice with Pten deletions in lymphocytes develop lymphadenopathy (due to a chronic polyclonal lymphoproliferative disorder) as well as CD4+ T lymphocytic lymphoma. The appearance of these diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether c-Myc is required for the development of these hematopoietic disorders in Pten-mutant mice, we generated inducible Pten and c-Myc double-knockout mice (Pten-/-c-Myc-/-). By comparing the hematopoietic phenotypes of the Pten-/-c-Myc-/- mice with those of Pten-mutant (Pten-/-) mice, we found that both sets of mice developed MPD and lymphadenopathy, however none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPD which developed in Pten-/- mice, which is dominated by granulocytes, megakaryocytes predominate in the MPD of Pten-/-c-Myc-/- mice. We have concluded that c-Myc is required for the development of both T lymphocytic lymphoma and the acute leukemic transformation of Pten-/- MPD, but is not essential for the development of chronic myeloid or lymphoid proliferative disorders. Our study suggests that deregulation of PI3K/Akt signaling in Pten-mutant hematopoietic cells protects these cells from apoptotic cell death, which results in chronic proliferative disorders, while the deregulation of c-Myc resulting from additional mutations promotes hematopoietic cell proliferation and blockage of maturation, and is absolutely required for the development of acute hematopoietic malignances. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting CXCR4 with cell-penetrating pepducins in lymphoma and lymphocytic leukemia

Blood ◽  
2012 ◽  
Vol 119 (7) ◽  
pp. 1717-1725 ◽  
Author(s):  
Katie O'Callaghan ◽  
Lydia Lee ◽  
Nga Nguyen ◽  
Mo-Ying Hsieh ◽  
Nicole C. Kaneider ◽  
...  
Keyword(s):  
Lymphocytic Leukemia ◽  
Response To Treatment ◽  
Treatment Concept ◽  
Hematologic Neoplasms ◽  
Cxcr4 Signaling ◽  
Lymphoid Malignancies ◽  
Cell Penetrating ◽  
Apoptotic Effect ◽  
New Treatment ◽  
Intracellular Loops

Abstract The chemokine receptor CXCR4, which normally regulates stromal stem cell interactions in the bone marrow, is highly expressed on a variety of malignant hematologic cells, including lymphoma and lymphocytic leukemias. A new treatment concept has arisen wherein CXCR4 may be an effective therapeutic target as an adjunct to treatment of hematologic neoplasms with chemo- and immunotherapy. In the present study, we developed pepducins, cell-penetrating lipopeptide antagonists of CXCR4, to interdict CXCL12-CXCR4 transmembrane signaling to intracellular G-proteins. We demonstrate that pepducins targeting the first (i1) or third (i3) intracellular loops of CXCR4 completely abrogate CXCL12-mediated cell migration of lymphocytic leukemias and lymphomas. Stromal-cell coculture protects lymphoma cells from apoptosis in response to treatment with the CD20-targeted Ab rituximab. However, combination treatment with CXCR4 pepducins and rituximab significantly increases the apoptotic effect of rituximab. Furthermore, treatment of mice bearing disseminated lymphoma xenografts with pepducins alone or in combination with rituximab significantly increased their survival. These data demonstrate that CXCL12-CXCR4 signaling can be effectively inhibited by cell-penetrating pepducins, which represents a potential new treatment strategy for lymphoid malignancies.

Chemotherapy With Infusion Related HLA-Mismatched Hematopoietic Stem Cells For Hematologic Malignancies: A Novel Therapeutic Strategy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5437-5437
Author(s):  
Li Fu ◽  
Zhao Wang
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Therapeutic Strategy ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Lymphocytic Leukemia ◽  
No Donor ◽  
Hematopoietic Stem ◽  
Novel Therapeutic

Abstract Objective To investigate the therapeutic outcomes of chemotherapy with infusion related HLA-mismatched hematopoietic stem cells for hematologic malignancies. Method Therapy responses and hematopoietic recovery as well as complications of 9 patients were analyzed. Result In the 9 patients aged 29-67 years, four were acute myeloid leukemia, one was acute B lymphocytic leukemia, two were multiple myeloma, one was Hodgkin’s disease, one was diffuse large B cell lymphoma. The average MNC was (3.12±1.29)×108/kg, CD34+ cells was (1.71± 1.00)×106/kg, CD3+ cells was (2.13±0.99)×108/kg. There was complete remission in four patients, partial remission in one, disease progression in four. Following up 2 to 14 months, four patients was in survival. No donor chimerism was detected and no graft-versus-host disease was observed in any patient. Conclusion Chemotherapy with infusion related HLA-mismatched hematopoietic stem cells for hematologic malignancies may provide a promising treatment method as a novel therapeutic strategy. Disclosures: No relevant conflicts of interest to declare.

All-Trans Retinoic Acid Synergizes With FLT3 Tyrosine Kinase Inhibition To Eliminate FLT3/ITD-Expressing Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3960-3960
Author(s):  
Hayley S Ma ◽  
Sarah M Greenblatt ◽  
Eric Jung ◽  
Amy S. Duffield ◽  
Li Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinoic Acid ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Apoptotic Cell ◽  
Leukemia Cells ◽  
Cure Rate ◽  
Hematopoietic Stem

Abstract FLT3 is one of the most frequently mutated genes in AML with approximately 1/3 of patients affected, and the internal tandem duplication (ITD) mutations portending a poor prognosis. To improve the cure rate for FLT3 mutant AML a number of FLT3 tyrosine kinase inhibitors (TKIs) have been developed to inhibit FLT3 signaling. While several recent FLT3 TKIs are proving increasingly successful at achieving high levels of FLT3 inhibition, there remain several problems in treating FLT3 mutant AML. One is that these drugs used as monotherapy achieve limited clinical responses and do not cure patients. While there is hope that combination with chemotherapy will achieve an improved cure rate in these patients without the necessity for hematopoietic stem cell transplantion (HSCT), the ultimate goal of studies are to achieve cures without chemotherapy, eliminating the short and long-term side effects that its use engenders. Thus, finding additional molecular targets that might synergize with FLT3 inhibition will move the field towards the goal of eliminating chemotherapy. One pathway known to play important roles in leukemia stem cell (LSC) survival and differentiation is the retinoic acid (RA) pathway. We therefore explored the combination of molecularly targeting the RA pathway together with FLT3 TKIs to determine the effect on FLT3 mutant leukemia cells. FLT3/ITD+ AML cell lines (Molm14 and MV411), along with FLT3/WT cell lines (THP-1, SEMK2 and NB4), were treated with FLT3 TKI alone (AC220 and sorafenib), ATRA alone, FLT3 TKI plus ATRA, or vehicle controls. Proliferative, apoptotic, cell-cycle and differentiation effects on the cells were assessed by MTT, annexin V binding, propidium iodide (PI) staining, CD11b staining and cell count assays. Highly synergistic effects were observed for the combination of ATRA with FLT3 TKIs against FLT3/ITD+ cells, with combination index (CI) values of 0.1-0.6. Colony forming unit (CFU) assays further demonstrated decreased clonogenicity of Molm14 and MV411 cells upon treatment with ATRA and sorafenib. A series of experiments were performed using a genetic model of spontaneous leukemia that we developed in which FLT3/ITD knock-in mice are bred with NUP98- HOXD13 (NHD13) transgenic mice, resulting in AML that is highly penetrant, lethal and transplantable. Cohorts of leukemic mice were generated by transplanting lineage negative (Lin-) BM cells from leukemic FLT3/ITD-NHD13 mice, and treated with either vehicle, sorafenib, ATRA or a combination of both drugs. We determined that treatment with sorafenib plus ATRA greatly decreases the level of engraftment at 2 and 8 weeks, and increases median survival, with some mice even cured of their disease. We also directly assessed the effect of the treatments (FLT3 TKI alone, ATRA alone, FLT3 TKI plus ATRA, or vehicle alone) on the proliferation of LSCs by using the different treatments on Lin- BM cells harvested from sick FLT3/ITD NHD13 mice ex vivo and found that ATRA further increased the anti-proliferative effect of FLT3 TKIs, with additive/synergistic CI values of 0.6-1. Lin- BM cells were also assessed in vitro in CFU assays of differentiation and clonogenicity, and combinatorial effects were observed. The results of these experiments show good synergy for this drug combination in vitro and improved survival/reduced LSC frequency in vivo. We believe these preclinical data are encouraging for the development of a clinical trial of ATRA plus FLT3 TKI in relapsed/refractory FLT3 mutant AML patients. Disclosures: No relevant conflicts of interest to declare.

Aspirin Induces Apoptosis in Human Leukemia Cells Independently of NF-κB and MAPKs through Alteration of the Mcl-1/Noxa Balance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4825-4825
Author(s):  
Ana M Cosialls ◽  
Daniel Iglesias-Serret ◽  
Maria Piqué ◽  
Montserrat Barragán ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Cell Types ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Protein Levels ◽  
Human Leukemia Cells ◽  
Induced Apoptosis

Abstract Abstract 4825 Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in most cell types. We examined the mechanism of aspirin-induced apoptosis in human leukemia cells. Our results show that aspirin induced apoptosis in leukemia Jurkat T cells independently of NF-κB. Although aspirin induced p38 MAPK and c-Jun N-terminal kinase (JNK) activation, selective inhibitors of these kinases did not inhibit aspirin-induced apoptosis. We studied the regulation of Bcl-2 family members in aspirin-induced apoptosis. The mRNA levels of some pro-apoptotic members, such as BIM, NOXA, BMF or PUMA, were induced by aspirin. However, none of these pro-apoptotic proteins increased and the levels of Mcl-1 protein were reduced. Interestingly, in the presence of aspirin the protein levels of Noxa remained high. This alteration of the Mcl-1/Noxa balance was also found in other leukemia cell lines and primary chronic lymphocytic leukemia cells (CLL). Furthermore, in CLL cells aspirin induced an increase in the protein levels of Noxa. Knockdown of Noxa or Puma significantly attenuated aspirin-induced apoptosis. These results indicate that aspirin induces apoptosis through alteration of the Mcl-1/Noxa balance. Disclosures No relevant conflicts of interest to declare.

Deacetylase Inhibitor Cocktails Provide Striking Synergistic Interactions in Human Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4404-4404
Author(s):  
Michele Cea ◽  
Antonia Cagnetta ◽  
Floriana Fruscione ◽  
Santina Bruzzone ◽  
Gabriele Zoppoli ◽  
...  
Keyword(s):  
Hydroxamic Acid ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia

Abstract Abstract 4404 Cancer cells almost invariably exhibit aberrant histone deacetylase (HDAC) activity leading to changes in chromatine structure, altered gene expression, poor differentiation, impaired apoptosis and increased proliferation. Accordingly, virtually all the HDAC inhibitors currently available show some degree of antitumor activity in preclinical cancer models and several of these compounds are currently under investigation or already approved for the treatment of human malignancies. Such is the case of the hydroxamic acid derivative suberoylanilide hydroxamic acid (Vorinostat, Zolinza), approved for the treatment of cutaneous T cell lymphomas. Sirtuins are a large family of deacetylases characterized by a unique, NAD+-dependent enzymatic mechanism. In addition to their established role in metabolism and longevity, recent evidence points to an emerging role for sirtuins in carcinogenesis. In the attempt to identify drug combinations that would increase the activity of traditional HDAC inhibitors we have explored the combination of valproic acid (VA) and butyrate (BU) with the sirtuin inhibitors cambinol and sirtinol in primary B-cell chronic lymphocytic leukemia (B-CLL) cells (n=35), acute myelogenous leukemia (AML) cells (n=10) and leukemia cell lines. Cell viability was assessed by propidium iodide staining and flow cytometry. Combination indices were determined using the median-effect method. In leukemia cells, exposure to sirtuin inhibitors synergistically increased VA and BU mediated cytotoxicity. Conversely, these drugs were poorly active and failed to show any cooperation in healthy cells, including peripheral blood mononuclear cells and fibroblasts, suggesting a cancer-specific mode of action. Similar results were obtained by combining VA or BU with the Nampt inhibitor APO866, which reduces intracellular NAD+ levels and thereby prevents sirtuin activity. Remarkably, SIRT1 and SIRT6 inhibition per se did not seem to account for cell demise upon HDAC inhibition since expression of a dominant negative SIRT1 isoform or RNA interference-mediated SIRT6 silencing failed to increase VA and BU activity. Our data indicate a specific requirement by leukemia cells for sustained sirtuin activity when classical HDACs are inhibited. This feature is suitable to be therapeutically exploited by combining sirtuin inhibitors or APO866 with classical HDAC inhibitors especially for the treatment of hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1209-1209
Author(s):  
Chun Shik Park ◽  
Koramit Suppipat ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

Re-Exposure of Cord Blood (CB) to Non-Inherited Maternal HLA Antigens (NIMA) Improves Transplant Outcome in Patients (pts) with Hematologic Malignancies and May Reduce Relapse.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 661-661
Author(s):  
Jon J van Rood ◽  
Cladd E Stevens ◽  
Jacqueline Smits ◽  
Carmelita Carrier ◽  
Carol Carpenter ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Hla Antigens ◽  
Human Leukocyte ◽  
Hematologic Malignancies ◽  
Hematopoietic Stem ◽  
Leukocyte Antigens ◽  
Grade Ii ◽  
The Impact ◽  
Related Mortality

Abstract Abstract 661 CB hematopoietic stem cell transplantation (CBT) can be successful even if donor and recipient are not fully matched for human leukocyte antigens (HLA). This may result, at least in part, from tolerance-inducing events during pregnancy, but this concept has not been tested to date. Hence we analyzed the impact of fetal exposure to NIMA of the HLA-A, -B antigens or -DRB1 alleles on the outcome of 1121 pts with hematologic malignancies. All pts received single CB units provided by the NYBC, for treatment of ALL (N=451), AML (N=376), CML (N=116), MDS (N=79), other (N=99); 22% were transplanted in advanced stage. Median age was 9.7 years (range: 0.1-67); 29% of recipients were >16 years. Most pts (96%) received myeloablative cytoreduction. Sixty-two pts received fully matched grafts while 1059 received units mismatched (MM) for one or two HLA antigens. Of these, 79 (7%) had a MM antigen which was identical to a donor NIMA (Example: Pt: A1, A3; CBU: A1, A2; mother-CBU: A1, A3; A3 is NIMA). NIMA match was found in 25 recipients with one HLA MM and 54 of those with two MM. The NIMA match was identified after the transplant and was not used in unit selection. In multivariate analyses, NIMA matched transplants (NMTs), showed faster neutrophil recovery (RR=1.3, p=0.043), even for grafts with cell dose <3×107 (RR=1.6, p=0.053). There was no difference in the incidence of acute (grade II-IV) or chronic GvHD. 3-year relapse risk (cumulative incidence 22%) was reduced compared to 1 or 2 HLA MM no NIMA matched transplants, especially in pts with myelogenous malignancies given units with 1 HLA MM (RR=0.2, p=0.074). Further, 3-year transplant-related mortality was reduced (RR=0.7, p=0.034), particularly in pts ≥5 years old (RR=0.5, p=0.006), as was the 3-year overall mortality (RR= 0.7, p=0.029 and RR=0.6, p=0.015, respectively). As a result, in the NMTs, treatment failure (relapse or death) was significantly lower, particularly in pts ≥5 years (RR=0.7, p=0.019) and DFS was significantly improved (figure) and was similar to that of the 0 HLA MM group. These findings are the first indication that donor exposure to NIMA can improve post-transplant survival in unrelated CBT and might reduce relapse. We propose to include the NIMA of CB units in search algorithms. Thus, for pts lacking fully HLA matched grafts, HLA MM but NIMA matched CB units could be selected preferentially, since no adverse effects were seen. This strategy of selecting HLA MM grafts with optimal outcome effectively “expands” the current CB Inventory several-fold.Patient GroupNRR(95% Cl)p value0 MM360.5(0.3–0.8)0.0051 MM / NIMA Match180.4(0.2–0.9)0.0262 MM / NIMA Match400.8(0.5–1.2)0.3091 MM / No NIMA Match229reference group2 MM / No NIMA Match4871.1(0.9–1.3)0.365 Disclosures: No relevant conflicts of interest to declare.

Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1372-1372
Author(s):  
Hendrik W. Van Deventer ◽  
Robert Mango ◽  
Jonathan Serody
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Mesenchymal Cells ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

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