Establishment of a Novel CNS Infiltrated Xenograft Model through Engraftment of Patient-Derived Acute Lymphoblastic Leukemic Cells Into NOD/SCID/γc Null Mouse

Abstract Abstract 3248 Background and Purpose: Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although improvements in treatment regimen have raised the 5-year survival rate as high as 80% for pediatric ALL patients, a minority of patients with various risk factors, including central nervous system (CNS) infiltration continue to have poor prognosis. Recently, bone marrow (BM) microenvironments which support leukemic stem cells have become noticed as an important element which can influence treatment response and relapse of the disease. Although leukemic cells appear to be completely eradicated through treatment, they are thought to survive within bone marrow and/or extramedullary microenvironments, such as CNS, causing disease recurrence. However, little is known about the CNS microenvironment for leukemic cells because of the lack of appropriate animal model. Even though several investigators have tried to establish a CNS infiltrated model of leukemia, major limitation with these studies are the use of leukemic cell lines and the preconditioning of recipient mice, which did not represent CNS leukemia observed in patients. Here we report the establishment of a novel xenograft model for primary human ALL using NOD/SCID/γc null (NOG) mouse. Without irradiation, this model recapitulates CNS as well as extramedullary leukemic infiltration (hereby referred to as the h-leukemic NOG model). Result: Primary bone marrow samples were collected from 9 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOG mice. Primary samples from 8 out of 9 patients were successfully engrafted. Engrafted leukemic cells could be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as 95% BM chimerism and showed that blast phenotypes were conserved through serial transplantations. Of note, extramedullary organs including the CNS, liver, spleen, and kidneys showed the leukemic invasion consistent with those of the donor ALL patients. Liver pathology in the h-leukemic NOG model is identical to that seen in the ALL patients. We also showed the existence of a functional niche in the liver mediated by SDF-1/CXCR4 axis. In terms of the CNS involvement, we observed the progressive infiltration of leukemic cells into the Virchow-Robin space that is consistent with the pathology of human ALL patients. Using this model, we examined the mechanism of dissemination and harboring of leukemic cells in the CNS niche. Discussion: NOG mice model for engraftment of human leukemic cells provides useful insights into the biology of ALL and allows us to answer various questions concerning the mechanism of extramedullary invasion and expansion. We have reported that NOG mice have significantly better human hematopoietic cell engraftment in the BM and extramedullary organs than other immunodeficient mice (Hiramatsu H. Blood. 2003), and is capable of supporting the growth of human neoplastic cells (Kato M. Nature. 2009). Here we report that this non-preconditioned mouse xenograft model reproduces leukemic extramedullary involvement, including the CNS, in sustaining leukemic cells. This approach provides a more sophisticated and physiological model suitable for the evaluation of molecular interactions between patient leukemic cells and host niche. Our h-leukemic NOG model will provide a powerful tool to analyze the CNS niche that harbors leukemia initiating cells. Moreover, this model would be a useful platform for developing novel anti-leukemic therapies that target CNS extramedullary niche. Disclosures: No relevant conflicts of interest to declare.

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Targeting the Leukemia-Associated Hypoxic Microenvironment with Hypoxia-Activated Prodrug PR-104

Blood ◽

10.1182/blood.v116.21.868.868 ◽

2010 ◽

Vol 116 (21) ◽

pp. 868-868

Author(s):

Juliana Benito ◽

Yuexi Shi ◽

Barbara Szymanska ◽

Hernan Carol ◽

Ingrid Bohem ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Leukemia Cell ◽

Chemotherapeutic Agents ◽

Leukemic Cells ◽

Immunodeficient Mice ◽

Hypoxic Conditions ◽

Nog Mice

Abstract Abstract 868 Interactions between leukemia cells and the bone marrow (BM) microenvironment are known to promote leukemia cell survival and confer resistance to drugs commonly used in the management of this disease. We investigated the protective role of hypoxia in the BM microenvironment. We observed a marked expansion of hypoxic niches in the bone marrow of immunodeficient mice engrafted with the acute lymphoblastic leukemia (ALL) cell line Nalm6 and with primary ALL cells, as detected by the reductive 2-nitroimidazole compound pimonidazole (PIM), which forms stable adducts in hypoxic regions. We further demonstrated induction of the chemokine receptor CXCR4, and of the enzyme carbonic anhydrase 9 (CAIX), both targets of Hypoxia-Inducible Factor 1α (HIF-1a), in hypoxic areas of BM from mice harboring the Nalm6 xenografts. Furthermore, we used a CML blast crisis model to evaluate the time course of hypoxia expansion in the BM. In C57Bl6/J mice engrafted with murine HSCs co-expressing BCR/ABL and Nup98 we observed a time-dependent increase in PIM positive areas which coincided with the presence of GFP positive cells. In line with these findings, HIF-1α was highly expressed in BM biopsies from newly diagnosed ALL patients (n=15) but was significantly reduced when the patients achieved complete remission (CR). Culture under hypoxic conditions (1% O2) conferred resistance of pre-B ALL cells REH, Nalm-6, and of AML OCI-AML3 leukemic cells against several chemotherapeutic agents including vincristine, methotrexate and idarubicin (% of Annexin V(+) cells at 21% vs 1%O2; REH plus 1ng/ml vincristine:88.5+/−2.3 vs 18.1+/−10.3; REH plus 0.25uM etoposide: 86.4 +/−7 vs 14.4+/−8.6; Nalm6 plus 10ng/ml methotrexate: 33.4+/−2.7 vs 4.6+/−1; OCI-AML3 plus 50ng/ml idarubicin: 28.45+/−5 vs 12.76+/−1) . Taken together, these results provide rationale for examining the potential of hypoxia-activated pro-drugs to eliminate leukemia progenitor cells within hypoxic niches. To this end, we tested the hypoxia-activated prodrug PR104, a dinitrobenzamide nitrogen mustard that is reduced to its active metabolites under hypoxic conditions (Patterson et al., Clin Can Res 2007). In vitro, PR-104 induced cell death in three different leukemia cell lines (Nalm6, REH and the AML line Molm13) selectively under hypoxic (pO2 1%) conditions. The anti-leukemic efficacy of PR-104 as a single agent was next examined in several in vivo leukemia models. Administration of PR-104 prolonged survival and decreased leukemia burden of 1) NOD/Scid/IL2Rg-KO (NOG) mice injected with cells from primary refractory FLT3-mutated AML; 2) NOG mice injected with leukemic cells from an infant with MLL-rearranged B-lineage ALL; and 3) NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice injected with Nalm6-luciferase ALL cells. Next, we evaluated anti-tumor effects of PR-104 at clinically relevant doses (200, 100 and 50 mg/kg) and at maximal tolerated dose (550 mg/kg) in two ALL xenograft models (a T-lineage ALL and a B-cell precursor ALL) (Figure 1, studies supported by NCI NO1CM42216 and by PPTP contract NO1-CM91001-03). Compared to vehicle control, PR-104 significantly delayed progression of the T-ALL xenografts at all doses tested and at three of the four doses (550, 200 and 100 mg/kg) of B-cell pre-ALL, which resulted in significantly increased event-free survival (EFS) of mice in the treatment groups. Altogether, these findings strongly suggest that targeting hypoxia is feasible. If successful, this approach may significantly impact leukemia therapy and ultimately improve patient survival. This concept is currently being tested in an ongoing Phase I clinical trial of PR-104 in relapsed/refractory AML patients. Disclosures: Wilson: PROACTA: Equity Ownership. Konopleva:PROACTA: PI on clin trial funded by Proacta.

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CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.

Blood ◽

10.1182/blood.v108.11.2542.2542 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2542-2542

Author(s):

Christoph Le Viseur ◽

Marc Hotfilder ◽

Annegret Rosemann ◽

Ronald Stam ◽

Andre Schrauder ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Current Data ◽

Scid Mice ◽

Functional Assay ◽

Childhood All ◽

Mouse Xenograft Model

Abstract Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.

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A Novel Small Molecule with Epigenetic Activity Prolongs Survival in a Mouse Model of Multiple Myeloma Refractory to Standard Drugs

Blood ◽

10.1182/blood.v118.21.5121.5121 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5121-5121

Author(s):

Sergei Vatolin ◽

Khan Nazeer Shahper ◽

Yvonne Parker ◽

Daniel Lindner ◽

Frederic J. Reu

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Conflicts Of Interest ◽

Chromatin Condensation ◽

Xenograft Model ◽

Myeloma Cells ◽

Tail Vein ◽

Tail Vein Injection ◽

Mouse Xenograft Model ◽

Number Of Patients

Abstract Abstract 5121 Multiple myeloma refractory to bortezomib, IMiDs™, and conventional therapies represents an unmet medical need. An increasing number of patients progress to this stage since treatment related mortality has decreased. To test promising compounds for activity in this setting we established an NSG mouse xenograft model with serial transplantation by tail vein injection of myeloma cells from a patient with IgG kappa myeloma relapsed and refractory to all standard drugs. Eight days after tail vein injection monoclonal human IgG can be detected in serum. Bone marrow engraftment in young (6–12 weeks) NSG mice after sublethal radiation (275cGy) is close to 100% (n=32). Untreated mice die within less than 2 months, usually with liver and spleen metastasis (anti-human CD138 flow cytometry). In a drug screen that used a novel method developed in our lab, chromatin condensation PCR, we identified a non nucleoside compound (4I3) that potently (1mM) reactivated expression of epigenetically silenced genes and displayed cancer-specific growth and survival inhibition in myeloma cell lines but not normal cells. Normal bone marrow cells continued to divide at doses 10x higher than required to kill 80% of myeloma cells. 4I3 suppressed DNMT1 protein but rapid cell kill (within 1–2 days) suggested additional mechanisms which we currently investigate. Given IV to mice after documentation of engraftment by IgG serum immunoblots, it prolonged survival in an ongoing experiment. Updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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Lineage Switch Under Blinatumomab of a Relapsed Common ALL Co-Expressing Myeloid Markers without MLL Rearrangement

Blood ◽

10.1182/blood.v128.22.5196.5196 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5196-5196

Author(s):

Annabel Zoghbi ◽

Beate Winkler ◽

Udo zur Stadt ◽

Ingo Müller ◽

Gabriele Escherich

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Lymphoblastic Leukemia ◽

Palliative Therapy ◽

Bone Marrow Aspiration ◽

Leukemic Cells ◽

Lineage Switch

Abstract Lineage switch under Blinatumomab of a relapsed Common ALL co-expressing myeloid markers without MLL rearrangement Annabel Zoghbi, Udo zur Stadt, Beate Winkler, Ingo Müller and Gabriele Escherich Background The bispecific antibody blinatumomab induces T cell-mediated B cell lysis by adhering to CD3 positive cytotoxic T cells and attaching to CD19, a marker which is commonly seen in lymphoblastic malignancies of the B cell lineage. It has been proven to be an effective therapeutic option for adult and pediatric refractory or relapsed B cell precursor acute lymphoblastic leukemia (ALL). Lineage switch from ALL to a CD19 negative myeloid phenotype is well known in patients with mixed lineage leukemia (MLL) gene rearrangements and has been recently described following blinatumomab treatment in an infant with MLL rearrangement as well as for two patients with MLL rearrangement who were treated with CAR-T cells. Case description A nine year old girl with common ALL ( strong positivity for CD 10,19, 24,34,45, 52 and 79a) with the coexpression of myeloid markers (CD13 86% and CD66c 56%) was treated according to CoALL 08-09 high risk protocol and showed a very slow MRD response. Cytogenetic studies showed a hyperdiploid karyotyp without evidence of a MLL, BCR/ABL or TEL/AML rearrangement. Only one month into maintenance therapy she relapsed with the phenotype of the initial blasts. Because of non-response (M3 marrow) after the first two cycles of treatment accorting to the IntReALL protocol with a M3 marrow. The patient became aplastic for 2 months and developed aspergillus pneumonia, which was treated by combination of antifungals and granulocyte infusions. Nevertherless, blinatumomab treatment was initiated. The patient tolerated treatment well and did not develop a cytokine release syndrome, but eventually recovered granulopoiesis under blinatumomab treatment. Leukemic blasts below the 1% border, however, were detectable throughout immunotherapy. In the absence of other opportunities, we planned on a HSCT and in the event of relapse thereafter re-initiation of Blinatumomab in the allogeneic setting with the new immune system developing. Hence, the patient underwent an uneventful MUD-HSCT in March 2016 following conditioning with TBI/VP-16 and serotherapy with alemtuzumab due to CD52 expression of the blasts. On day 29 after transplant, a routine bone marrow aspiration revealed a second molecular relapse at the order of 2 x 10-3with the same immunophenotype. Thus, immunosuppression with cyclosporine A was drastically lowered and a grade II skin GvHD developed, which was controlled by topic treatment, before CsA was discontinued. Blinatumomab was reinitiated on day 49 after SCT. After two and a half weeks on therapy, an increase in LDH and leukocytes was visible. Bone marrow aspiration showed disease progression with 70% blasts, which now were CD19 negative with the immunophenotypical and morphological attributes of myeloid leukemia. Nevertheless, initial MRD markers still were strongly positive, indicating the common precursor with the initial phenotype. Detailed analysis will be presented. Cytogenetic analysis of the relapse samples revealed no changes. With no curative approach remaining, palliative therapy with cytarabine and thioguanine was initiated which could not control the fulminant expansion of leukemic cells, leading to the patient's death within four days from diagnosis. Conclusion CD19 negative relapses following blinatumomab therapy have been perceived in recent treatment studies, while reports on immunologic and morphologic presentation of a myeloid phenotype are scarce. Our patient coexpressed myeloid markers, CD13 and CD66c, from the start, thus, it is possible that subclones who did not carry CD19 had a pronounced selection advantage during blinatumomab treatment, leading to their vast expansion. Disclosures No relevant conflicts of interest to declare.

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In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Blood ◽

10.1182/blood.v124.21.1020.1020 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1020-1020

Author(s):

Hiromichi Matsushita ◽

Takashi Yahata ◽

Yin Sheng ◽

Yoshihiko Nakamura ◽

Yukari Muguruma ◽

...

Keyword(s):

Cord Blood ◽

Conflicts Of Interest ◽

Nuclear Bodies ◽

Leukemic Cells ◽

Human Cord Blood ◽

Immunodeficient Mice ◽

Cd34 Cells ◽

Nog Mice

Abstract Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. Disclosures No relevant conflicts of interest to declare.

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Disturbed CXCR4/CXCL12 Axis In Pediatric Precursor B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.2643.2643 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2643-2643 ◽

Cited By ~ 1

Author(s):

Lieke C.J. van den Berk ◽

Arian van der Veer ◽

Marieke E. Willemse ◽

Myrte J.G.A. Theeuwes ◽

Mirjam W. Luijendijk ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Serum Levels ◽

Initial Diagnosis ◽

Leukemic Cells ◽

Cxcr4 Receptor ◽

Cell Acute Lymphoblastic Leukemia

Abstract Malignant cells that infiltrate the bone marrow (BM) interfere with the normal cellular behavior of supporting cells, thereby creating an alternative malignant niche. This intercellular communication is mostly mediated by cytokines and their receptors. In this study, we find that expression of the CXCR4 receptor is significantly increased in pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) cells compared with normal mononuclear hematopoietic cells derived of the bone marrow (p=0.016). Furthermore, we show that high CXCR4 expression is correlated with an unfavorable clinical outcome in BCP-ALL (5-yr CIR ±SE: 38.4% ±6.9% in CXCR4-high versus 12.0% ±4.6% in CXCR4-low expressing patients, p<0.001). Interestingly, BM serum levels of the CXCR4 ligand (CXCL12) are 2.7-fold lower (p=0.005) in samples taken at initial diagnosis of BCP-ALL compared with the levels in samples taken of non-leukemic controls. We show that induction chemotherapy restores CXCL12 levels in the BM to normal levels. Blocking the CXCR4 receptor with Plerixafor (FDA-approved drug) showed that the lower CXCL12 serum levels at initial diagnosis could not be explained by consumption by the leukemic cells, nor did we observe an altered CXCL12-production capacity of BM-MSC at this time-point. We rather observed that a very high density of leukemic cells negatively affected CXCL12 production by the BM-MSC while stimulating the secretion levels of G-CSF. These results suggest that highly proliferative leukemic cells are able to down-regulate the production of cytokines involved in homing (CXCL12), while simultaneously up-regulating the production of cytokines involved in hematopoietic mobilization (G-CSF). This disbalance may stimulate the spreading of BCP-ALL outside the BM. The data presented here suggest that interference with the CXCR4/CXCL12 axis (for instance by using Plerixafor) may be an effective way to mobilize BCP-ALL cells; the more ALL cells become mobilized, the less ALL cells may escape from combination chemotherapy. In proof-of concept studies, this hypothesis needs to be validated to pave the way for implementation in future treatment protocols for children with ALL. Disclosures: No relevant conflicts of interest to declare.

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B-Cell Precursor Acute Lymphoblastic Leukemia Cells Create a Self-Reinforcing Niche Independent of the CXCR4/CXCL12 Axis

Blood ◽

10.1182/blood.v126.23.1297.1297 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1297-1297

Author(s):

Bob de Rooij ◽

Roel Polak ◽

Rob Pieters ◽

Monique L. Den Boer

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Hematopoietic Cells ◽

Leukemic Cells ◽

Cell Precursor ◽

Cxcr4 Antagonist

Abstract Background Acute lymphoblastic leukemia (ALL) cells create a leukemic niche that protects malignant cells from the effects of cytostatic agents and immune cells by altering their bone marrow microenvironment. This malignant process can be counteracted by impairing the homing of leukemic cells towards the bone marrow. Hematopoietic cells express the chemokine receptor CXCR4 and migrate towards its ligand CXCL12, which is actively produced by MSCs in the bone marrow. Therefore clinical trials have been initiated using the CXCR4 antagonist AMD3100 (Plerixafor) during leukemia treatment. However, these trials, as well as priming of AML in more than 4000 patients using a CXCR4 dependent mechanism, have not resulted in improved overall survival rates. This suggests that CXCR4 inhibition is not sufficient to disrupt leukemic niches. Objectives In this study we investigated how leukemic cells regulate the chemoattractive properties of their microenvironment. Results Here we show, using an ex vivo niche model with primary MSCs, that B-cell precursor ALL (BCP-ALL) cells affect their healthy microenvironment without altering CXCL12 secretion. Using a transwell migration assay we studied the chemoattractive properties and chemokine secretion patterns of several cell types and co-cultures. We confirmed that BCP-ALL cells migrate towards a CXCL12 gradient produced by primary MSCs (11-fold more migrated cells compared to background, p < 0.001). Inhibition of CXCR4 by AMD3100 reduced migration towards MSCs by 80% (p < 0.01). BCP-ALL cells migrated even more towards co-cultures of BCP-ALL cells and primary MSCs (24-fold more migrated cells compared to background, p < 0.001). Strikingly, this ex vivo leukemic niche did not produce higher levels of CXCL12 compared to MSC mono-cultures. Moreover, the induced migration towards MSC-ALL co-cultures could not be inhibited by AMD3100 treatment, indicating that BCP-ALL cells enhance the chemoattractive properties of their microenvironment in a CXCL12-independent manner. In contrast to BCP-ALL cells, the migration of CD34+ progenitor cells towards co-cultures of BCP-ALL cells and MSCs was significantly reduced (0.8-fold compared to migration towards MSCs, p < 0.05). Similar results were observed when we studied the migratory behavior of MSCs. MSCs actively migrated towards BCP-ALL cells (1.7 fold compared to background, p < 0.001), while migration of MSCs was significantly reduced towards MSC-ALL co-cultures (0.4-fold compared to migration towards BCP-ALL, p < 0.001). To find candidate factors influencing this process, we quantified the secreted levels of 64 cytokines in co-cultures of patient-derived BCP-ALL cells and MSCs. We observed leukemia-driven cytokine secretion patterns that were not influenced by the source of primary MSCs. In contrast to unaltered levels of CXCL12, we observed significant inductions of MCP-1/CCL2 and MDC/CCL22 (CCR4-ligands), IL8 and GRO-1 (CXCR1/2-ligands) and IP10/CXCL10 (CXCR3-ligands). Conclusion Our data indicate that leukemic cells alter the chemoattractive properties of their microenvironment, resulting in the secretion of multiple chemokines into the leukemic niche. This leukemic niche is highly potent in attracting BCP-ALL cells and repels the influx of healthy hematopoietic cells and MSCs using a CXCL12-independent mechanism. Furthermore, our results identify candidate factors that might be valuable future therapeutic targets. Disclosures No relevant conflicts of interest to declare.

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Trephine Immuno-Histochemistry As an Useful Adjunct in Characterizing Myelonecrosis in Resource Poor Settings - a Study from India

Blood ◽

10.1182/blood-2019-128772 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5028-5028

Author(s):

Shruti Mishra ◽

Somanath Padhi ◽

Rasheeda Mohamedali ◽

Kishore Kumar

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Hematologic Malignancy ◽

Lymphoblastic Leukemia ◽

Care Center ◽

Tertiary Care ◽

Specific Antigen ◽

Bone Marrow Necrosis ◽

Resource Poor ◽

Immuno Histochemistry

Introduction: Myelonecrosis is defined as necrosis of myeloid tissue within the hematopoietic medullary spaces with preservation of bony trabeculae. It is a rare finding on trephine which is reported to occur in 0.37% to 6.5% of all cases [1]. Most common cause of myelonecrosis is associated neoplasia [2]. Extensive myelonecrosis can make bone marrow aspiration and further evaluation difficult, leading to delay in diagnosis and management. We describe five such interesting cases with varying degree of myelonecrosis where immune-histochemistry in the necrosed zone helped us clinch the diagnosis. Cases and methods: Clinicopathological characteristics, bone marrow morphology and Immuno-histochemistry findings are tabulated below. In all the cases trephine biopsies were fixed with 10% neutral buffered formalin and decalcification was done with 14% EDTA for 48-72hrs (as per ICSH 2008 Guidelines). In all these cases, myelonecrosis was graded and ranged from mild to moderate. The procedure used was that of a semi-quantitative one. Grade I (mild): <20% of the biopsy Grade II (moderate-intermediate): 20-50% of the biopsy Grade III (severe-extensive): >50% of the biopsy. Gomori's technique was done for reticulin staining. Fibrosis was graded as per WHO protocols. Immuno-histochemistry was done by peroxidase - anti-peroxidase technique. Immuno-histochemical findings were recorded separately by two hemato-pathologists. Expression of each marker was graded as per strength and compared with the expression in the viable zones. The gradation of expression was done in a semi-quantitative manner. Mild expression: + Moderate expression: ++ Strong expression: +++ Discussion: Out of the five cases, three were hematological malignancies and rest two are metastatic solid tumors. Myelonecrosis in all the cases was of coagulative type. Myelonecrosis was extensive in 1/5 cases, moderate in 3/5 cases and patchy or focal in 1/5 cases. We found that the necrosed tumor cells retained their antigenicity for most of the markers. It was observed that even the intensity of expression was at par with that of the viable zones. The necrosed zone in case of B-cell precursor- acute lymphoblastic leukemia showed diffuse and strong positivity for CD10, CD79a, TdT and dim positive for CD34, thus confirming the diagnosis. Similarly, in the case of Non-Hodgkin's lymphoma the expression of markers in the necrosed areas showed strong antigenic expression for CD20, CD79a and nuclear positivity for Bcl 2, Bcl 6 and Mib 1. In the myeloma case retention of CD138, CD56 and Lambda light chain restriction was noted. Interestingly, both the cases with metastasis presented with bony pain and increased ALP (Alkaline Phosphatase). While in one case associated increase in PSA (Prostate specific antigen) in serum indicated the possibility of metastasis from a prostatic primary, the other case was evaluated in the line of myeloma. Immuno-histochemistry in the trephine suggested a possibility of metastasis from an occult primary. Later, on further evaluation a primary was found in the right breast of the patient. In both the metastatic cases, the expression of Cytokeratin was strong and diffuse. PSA was expressed in dot-like pattern. Jinkala et. al(3). in their study found that malignancy was the commonest cause of myelonecrosis. They found that neoplastic aetiology in 91% cases of marrow necrosis, out of which primary hematologic malignancy constituted 60% and rest was due to metastatic solid tumours. Also, most of the studies have proved that myelonecrosis itself is a bad prognostic factor in patients. Conclusion: Thus, we conclude Immuno-histochemistry is an useful adjunct to demonstrate the retained antigenicity in myelo-necrosed tissues; and could be useful in these cases as they are usually associated with scant and difficult aspirate. It can be done easily leading to a faster diagnosis of the patient even in resource poor settings too. This will save the patient from undergoing a painful bone marrow procedure again. References: 1. Markovic SN,. Pancytopenia due to bone marrow necrosis in acute myeloid leukemia: role of reactive CD8 cells. Am J Hematol 1998; 59:74. 2. Dunn Bone marrow necrosis in 38 adult cancer patients. J Famos Med Assoc 1993;92(12): 1107-10. 3. Jinkala S.R. Myelonecrosis: A Clinicopathological Study from a Tertiary Care Center in South India over a Twelve-Year Period. Bone Marrow Research 2014; 5 Figure Disclosures No relevant conflicts of interest to declare.

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Anti-Leukemic Activity of Mitochondrially-Directed Asymmetrical 2-2'-Binaphthoquinones.

Blood ◽

10.1182/blood.v114.22.4799.4799 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4799-4799

Author(s):

Rebecca Ricklis ◽

Milena Vuica-Ross ◽

Robert Brodsky ◽

Ed Sausville ◽

Chi Van Dang ◽

...

Keyword(s):

Cell Lines ◽

Mitochondrial Membrane ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Xenograft Model ◽

Leukemic Cells ◽

Rhodamine 123 ◽

A Genome ◽

Ic50 Values

Abstract Abstract 4799 Introduction Binaphthoquinones are unique molecules consisting of two linked naphthoquinone units. Previously, we regiospecifically synthesized a series of asymmetrical 2,2'-binaphthoquinones, which possess HIV integrase inhibitory activity. Potent activity against non-HIV-infected CEM-T4 acute lymphoblastic leukemia cells prompted us to investigate other cytotoxic mechanisms of these compounds. A genome-wide yeast screen uncovered that mitochondrial-related genes are required for sensitivity and resistance to these agents. Furthermore, by generating reactive oxygen species (ROS), biquinones halted yeast growth which was rescued by addition of N-acetylcysteine. Therefore, as part of our efforts to identify new compounds with anti-leukemic activity, we hypothesized that 2,2'-binaphthoquinones would be able to kill leukemic cells by interference with mitochondrial function. The majority of studies on antineoplastic effects of quinones have focused on benzoquinones, anthraquinones or monomeric naphthoquinones. However, nothing is published on the anti-leukemic action of asymmetrical 2,2'-binaphthoquinones, in which two different mononaphthoquinones are attached at the quinone double bond. Biquinones possess four carbonyl groups that have the potential to generate a greater number of ROS per one mole of quinone, and thereby cause more effective oxidative stress, than their monoquinone counterparts. In addition, the potential differences in cytotoxicities between monomeric and dimeric naphthoquinones result from many parameters, including the number of units, planarity, and the specific nature and pattern of aromatic functional groups. Materials and Methods Seventeen of our biquinones were dissolved in DMSO and incubated with four leukemia cell lines grown under standard conditions. All cultures were maintained with a constant DMSO concentration. A standard MTT cell proliferation assay was used to identify IC50 values. MV411 cells were incubated for 42-49 hours (2 nights) with two of the most potent compounds, biquinone #7 (BiQ7) and biquinone #10 (BiQ10), and they were assayed and analyzed by flow cytometry for loss of mitochondrial membrane potential (via rhodamine 123 staining), as well as a variety of apoptosis markers such as exposed phosphatidylserine, activated caspases, and sub-2n DNA increases. Simultaneously, preliminary toxicology experiments have been performed on mice. Results The IC50 values for both compounds were less than 5 micromolar (μM) against all four cell lines. For subsequent experiments, the cells were treated with either 5 μM BiQ7 or 5 μM BiQ10. A loss of mitochondrial membrane electrochemical potential, as expressed and evidenced by substantial decrease in fluorescence intensity of Rhodamine 123, was observed in 98-100% of the treated cells compared to control (5%). Loss of plasma membrane phosphatidylserine asymmetry was observed via Annexin V- PE (with 7AAD) staining in 94-95% of the treated cells and in only 18% of the control cells. Furthermore, the treated cells showed increases in staining for activated caspases 3 & 7 (BiQs 96-98%; control 10%). Finally, the cell cycle analyses via propidium iodide showed increases in sub-2n DNA in the treated cells compared to the control (control 12%; BiQ7 34%; and BiQ10 54%). Importantly, the preliminary toxicology results in mice suggest selectivity for neoplastic cells since no cytopenia or obvious end organ toxicity was observed with injection of 600 μM binaphthoquinones intraperitoneally daily for three weeks. Conclusion Our study shows that this new class of compounds possesses promising in vitro anti-leukemic effects by targeting mitochondrial membrane permeabilization, which occurs early in the apoptotic program and is located downstream of most identified chemotherapy resistance mechanisms in hematologic malignancies. In vivo experiments in xenograft model and ex vivo experiments on primary human cells are planned. Disclosures: No relevant conflicts of interest to declare.

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