Adhesion to Bone Marrow Stroma Inhibits Apoptosis of Chronic Lymphocytic Leukemia Cells

Abstract Abstract 359 Chromosomal translocations involving c-Myc are frequently found in high grade lymphoma and multiple myeloma. In contrast, c-Myc translocations rarely occur in low-grade lymphomas/leukemias like chronic lymphocytic leukemia (CLL), but when present they are associated with rapid disease progression and bad prognosis. Overexpression of c-myc may also be the result of increased transcription by several proto-oncogene transcription factors, including NF-kB. Mice with c-Myc de-regulation at different stages of B cell development develop either aggressive B cells lymphomas or plasma cell neoplasm. So far, no c-Myc mouse model developed low-grade lymphoma/leukemia. iMycCa mice develop an expansion of CD5+ peritoneal B1 cells, as compared with WT littermates mice. These mice have a normal life-span and very rarely develop B cell lymphoma at older age. Interestingly, in iMycCa mice mature B cells, but not plasma cells,could be rescued from apoptosis by administration of B cell-activating factor belonging to the TNF family (BAFF). To our surprise, double transgenic iMycCa/Baff-Tg (Myc/Baff) mice developed a disease resembling human CLL, with dramatically shorter mean survival than parental strains, due to early onset and rapid clonal expansion of a mature CD5+B220low B cell population. Those cells transferred the disease into Baff-Tg (Baff) mice with marked infiltration in lymphoid organs and bone marrow. Gene-expression analyses revealed that among the genes altered in Myc/Baff CD5+B220lowleukemia cells were those with known relevance to human CLL disease, including elevated anti-apoptotic Bcl2 family members. Apart from studies on individual genes, sub-network analysis was performed which showed enrichment of apoptosis-related and stress-induced gene sets in Myc/Baff CD5+CD3- leukemia cells. The NF-kB gene set, a major target downstream of BAFF signaling, was also enriched in Myc/Baff CD5+CD3- leukemia cells. We observed a continuum in levels of c-MYC mRNA in 166 samples using Affymetrix array analyses. Changes in c-Myc protein expression were confirmed by immunoblot analyses and correlated with disease progression. In accordance with the functions of c-Myc as a promoter of cell cycle progression, as well as apoptosis, we found enhanced spontaneous cell death in vitro in CLL cells expressing high levels of c-Myc, which could be abrogated by co culture with BAFF expressing nurse-like cells (NLC) or recombinant BAFF. In addition to its anti-apoptotic role, BAFF treatment of primary human CLL cells led to dramatically enhanced expression of c-Myc through the IKK/NF-kB pathway. Inhibition of the NF-kB pathway significantly reduced viability of both Myc/Baff CD5+CD3- leukemia cells and human CLL cells co-cultured with NLC. Also it significantly lowered CD5+B220low leukemia cell population in blood and spleen, and prevented the infiltration of leukemia cells into lymph nodes and bone marrow of transplanted mice. This study demonstrates a potential pathologic role for c-Myc, in the pathogenesis and progression of CLL. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v116.21.1372.1372 ◽

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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The Novel Phospholipid Gemcitabine Conjugate KPC34 Has Activity In Acute Leukemia

Blood ◽

10.1182/blood.v122.21.1425.1425 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1425-1425

Author(s):

Timothy Pardee ◽

Peter Alexander ◽

Kristin Stadelman ◽

Greg Kucera

Keyword(s):

Bone Marrow ◽

Survival Rate ◽

Leukemic Cell ◽

Leukemia Cells ◽

P Value ◽

Bone Marrow Stroma ◽

Acute Leukemias ◽

Leukemic Cell Lines ◽

Marrow Stroma ◽

Nm Range

Abstract Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are cancers of the bone marrow that in adults, occur mainly in the elderly. About 19,000 Americans develop these diseases annually and the 5 year survival rate is 30-40%. The prognosis for elderly patients is even worse with a 5 year survival rate of 10%. Treatment for AML and ALL involves induction chemotherapy using the deoxycytidine analog cytarabine followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Recurrent disease is much more difficult to treat successfully and elderly patients often have trouble tolerating repeated chemotherapy treatments. Chemoresistance can occur by several mechanisms including reduced activation of cytarabine via downregulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. Low expression of both dCK and ENT-1 correlate with poor prognosis in patients with acute leukemias. Another contributor to cytarabine resistance is the interaction of leukemia cells with bone marrow stroma. This interaction confers protection to leukemia cells from cytarabine by inhibiting apoptosis and ENT-1 function. Additionally, activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms α, β1, and β2 have been detected in both AML and ALL and promote leukemic proliferation and survival by activation of ERK and the anti-apoptotic protein BCL-2. The development of novel therapeutic agents or modifying existing drugs may be able to overcome chemoresistance in AML and ALL and improve clinical outcomes. KPC34 is a novel phospholipid conjugate of the deoxycytidine analog gemcitabine. Gemcitabine acts similarly to cytarabine and is administered IV to treat various tumors and lymphomas, but is rarely used for leukemias. Compared to cytarabine, gemcitabine treatment inhibits DNA replication more effectively, prevents repair of damaged DNA, and uniquely inhibits ribonucleotide reductase. KPC34's novel properties result in improved pharmacokinetics compared to gemcitabine and may be able to overcome leukemic chemoresistance to cytarabine by bypassing the need for ENT-1 uptake and dCK activation. In addition, KPC34 can be administered orally, is predicted to cross the blood-brain barrier, allowing it to target CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety is predicted to inhibit the activity of PKC. In order to determine if KPC34 has activity against acute leukemias we tested it against a variety of human and murine leukemic cell lines in vitro. KPC34 was highly cytotoxic to with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). Exposure of leukemia cells to KPC34 resulted in the induction of apoptosis as assessed by annexin V and PI assays. Human leukemic cell lines treated with KPC34 had dose-dependent reduction of colony formation, where normal donor hematopoietic stem cells did not. To test the ability of KPC34 to overcome stroma-mediated chemoresistance, SUPB15 cells were incubated in the presence or absence of a bone marrow stroma cell line for 48 hrs. The cells were then treated with 10 nM cytarabine or KPC-34. No significant difference was seen between either treatment without stroma, while KPC34 significantly increased apoptosis in the presence of stroma (p value ≤ 0.001, by Student’s t test). In order to assess the activity of KPC34 in vivo, we injected C57/BL6 mice with a syngeneic, murine Ph+ pre-B cell ALL, luciferase expressing cell line. When engraftment was established by bioluminescence imaging, mice were treated with 25 mg/kg KPC34 or water by oral gavage on days 1, 4 and 7. Treatment resulted in a significant prolongation of survival with treated mice having a median survival of 26 days compared to 13 days for controls (p value of 0.0042, by log-rank test). These data demonstrate the activity of KPC34 in multiple preclinical models of leukemia and support its continued translation to the clinic. Disclosures: Kucera: Wake Forest Baptist Health: KPC 34 Patent, KPC 34 Patent Patents & Royalties.

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