ALBERT

Description: Glucocorticoids (GCs) are used in combination chemotherapies as front-line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although effective, many patients relapse and become resistant to chemotherapy and GCs in particular. Why these patients relapse is not clear. We took a comprehensive, functional genomics approach to identify sources of GC resistance. A genome-wide shRNA screen identified the transcriptional coactivators EHMT2, EHMT1, and CBX3 as important contributors to GC-induced cell death. This complex selectively supports GC-induced expression of genes contributing to cell death. A metaanalysis of gene expression data from B-ALL patient specimens revealed that Aurora kinase B (AURKB), which restrains GC signaling by phosphorylating EHMT1-2, is overexpressed in relapsed B-ALL, suggesting it as a potential contributor to relapse. Inhibition of AURKB enhanced GC-induced expression of cell death genes, resulting in potentiation of GC cytotoxicity in cell lines and relapsed B-ALL patient samples. This function for AURKB is distinct from its canonical role in the cell cycle. These results show the utility of functional genomics in understanding mechanisms of resistance and rapidly identifying combination chemotherapeutics.

Description: Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p

Description: Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), 20% of children relapse with high death rates, so that new treatment modalities are needed. Recent studies have demonstrated that survivin, a member of the inhibitor of apoptosis (IAP) family proteins, is upregulated in ALL of relapsed patients but not in drug-sensitive ALL. The expression of survivin depends on the formation of a complex between β-catenin and its co-activator CBP. Selective suppression of CBP/β-catenin signaling using the novel small-molecule inhibitor ICG-001 offers the opportunity to sensitize leukemia cells to conventional treatment. We hypothesize that inhibition of CBP/β-catenin signaling by combining ICG-001 with conventional therapy represents a promising therapeutic principle to eradicate drug resistant ALL. To test this hypothesis, we used a NOD/SCID xenograft model engrafted with drug-resistant human pre-B ALL leukemia cells (1x106 cells/mouse) to first model the outcome of the patient in vivo. When human CD45 engraftment of 1% was detected by flow cytometry on day 26 post-leukemia-injection, VDL (Vincristine, Dexamethasone, L-Asparaginase) (n=7) or with saline as control (n=7) was administered for 4 weeks intraperitoneally (i.p.). Without treatment, all mice died between days 31–38 post-treatment with a median survival time (MST) of 36 days. In contrast, one animal of the VDL group died at day 14 post-treatment, the remaining 6 mice between days 67–77 post-treatment (MST=70 days, p

Description: Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor. Congenital defects in pre-B cell receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell receptor signaling represents a critical target for malignant transformation towards ALL. Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1-gene rearrangement are –like normal pre-B cells- highly selected for the expression of a functional pre-B cell receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell receptor resulted in a strong Ca2+ signal. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1, 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ-heavy chain locus encoding the central component of the pre-B cell receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ-chain was amplified, no pre-B cell receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell receptor signaling enables (E2A-PBX1; Type I) or suppresses (BCR-ABL1, MLL-AF4, Hyperdiploid; Type II) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1-induced Type I ALL cells (active pre-B cell receptor signaling) and MLL-AF4-induced Type II ALL cells (lack of pre-B cell receptor expression) with the BCR-ABL1 oncogene. Whereas growth of pre-B cell receptor-negative Type II ALL cells was accelerated by BCR-ABL1-transformation, pre-B cell receptor-positive Type I ALL cells rapidly eliminated by apoptosis within 9 days after BCR-ABL1-transduction. To identify factors that distinguish Type I (E2A-PBX1) and Type II (BCR-ABL1, MLLAF4, Hyperdiploid) ALL and that may explain the divergent role of pre-B cell receptor signaling in these groups, we performed a comparative gene expression including a metaanalysis of published microarray data and quantitative RT-PCR. Compared to E2A-PBX1 Type I ALL, MYC mRNA levels are on average 4-fold, 6-fold and 2.5-fold higher in BCR-ABL1, MLL-AF4 and Hyperdiploid ALL cells, respectively. To test whether high expression levels of MYC render leukemia cells non-permissive to pre-B cell receptor expression, we studied bone marrow B cell precursors from Rag2−/− mice that carry a tetracycline-inducible μ-chain transgene (Hess et al., 2001) as cell culture model for inducible pre-B cell receptor expression. When expression of the pre-B cell receptor was induced in normal IL7-dependent B cell precursors, the cells were induced to first proliferate and subsequently differentiate, hence mirroring normal stages of early B cell development. We then transformed Rag2−/− Tet-μ-chain B cell precursors by retroviral transduction with MYC. MYC-transformed cells rapidly outcompeted untransduced normal IL7-dependent B cell precursors in cell culture. Induction of pre-B cell receptor expression, however, completely reversed growth kinetics and within a few days, normal untransduced pre-B cell receptor-positive cells had a substantial growth advantage over MYC-transduced pre-B cell receptor-positive cells that were progressively lost in cell culture. Interestingly, this growth pattern was reversible by subsequent ablation of pre-B cell receptor expression: After Tet-mediated ablation of pre-B cell receptor expression, the initial growth kinetics were restored and MYC-transduced pre-B cell receptor-negative cells regained a substantial growth advantage. These findings demonstrate that different levels of MYC expression determine permissiveness of ALL cells for pre-B cell receptor signaling. Hence, the pre-B cell receptor suppresses outgrowth of Type II leukemia by censoring high levels of MYC expression.

Description: Key Points We evaluated interference with integrin alpha4–mediated stromal adhesion as a new acute lymphoblastic leukemia treatment. Integrin alpha4 blockade using natalizumab in combination with chemotherapy sensitizes pre-B acute lymphoblastic leukemia to chemotherapy.

Description: Abstract 274 Background: The E2A-PBX1 [t(1;19)(q23;p13)] fusion is found in ≂f4% of cases of childhood ALL and involves a rearrangement of the TCF3 gene (encoding the E2A transcription factor). TCF3 (E2A) is not only a critical regulator of B cell lineage commitment and early B cell development (Müschen et al., 2002; Sigvardsson et al., 2002), it also cooperates with LEF1 to activate canonical WNT/β-catenin signaling (Hovanes et al., 2001; Merrill et al., 2001). Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor. Congenital defects in pre-B cell receptor-related signaling molecules cause a severe differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at an early pre-B cell stage in the vast majority of cases. Given that the pre-B cell receptor drives both proliferation and differentiation of normal B cell precursors, we test here the hypothesis that pre-B cell receptor signaling represents a critical target for malignant transformation towards ALL. Results: Studying 148 cases of pre-B cell-derived human ALL, we found that pre-B cell receptor expression and function is linked to specific cytogenetic subgroups: ALL cells carrying an E2A-PBX1-gene rearrangement are, like normal pre-B cells, highly selected for the expression of a functional pre-B cell receptor. In all 8 ALL cases with E2A-PBX1 fusion, engagement of the pre-B cell receptor resulted in a strong Ca2+ signal, which strongly suggests that E2A-PBX1 leukemia clones are selected for active pre-B cell receptor signaling. In striking contrast, ALL cells carrying BCR-ABL1- or MLL-AF4 fusion genes and ALL cells with hyperdiploid karyotype lack expression of a functional pre-B cell receptor in virtually all cases. Only 10 of 57 cases with BCR-ABL1, 0 of 7 cases with MLL-AF4 and 1 of 30 cases with hyperdiploid karyotype a productively rearranged μ -heavy chain locus encoding the central component of the pre-B cell receptor, was found. Even in the few BCR-ABL1 ALL cases, in which a productively rearranged μ -chain was amplified, no pre-B cell receptor was expressed. Based on these findings, we hypothesize that ALL can be subdivided into two groups based on whether pre-B cell receptor signaling enables (E2A-PBX1) or suppresses (BCR-ABL1, MLL-AF4, Hyperdiploid, likely other subtypes) leukemic growth. In a proof-of-concept experiment, we super-transformed E2A-PBX1-induced ALL cells (active pre-B cell receptor signaling) and MLL-AF4-ALL cells lacking pre-B cell receptor function with the BCR-ABL1 oncogene. The BCR-ABL1 oncogene was chosen, because it is only found in leukemia cells that lack pre-B cell receptor function. Whereas growth of pre-B cell receptor-negative MLL-AF4 ALL cells was strongly accelerated by BCR-ABL1-transformation, pre-B cell receptor-positive E2A-PBX1 ALL cells were rapidly eliminated within 9 days after BCR-ABL1-transduction. Interestingly, incubation of E2A-PBX1 ALL cells survived BCR-ABL1-transduction only in the presence of 10 μ mol/l of the BCR-ABL1 kinase inhibitor Imatinib. To identify factors that distinguish E2A-PBX1 and other ALL subtypes (BCR-ABL1, MLL-AF4, Hyperdiploid) that may explain the divergent role of pre-B cell receptor signaling in these groups, we performed a comparative gene expression including a meta-analysis of published microarray data and quantitative RT-PCR. In this analysis, E2A-PBX1 ALL cells were distinguished by high expression levels of pre-B cell receptor-related signaling molecules (e.g. BLNK, SYK, BTK). The most prominent gene expression differences involve canonical WNT/β-catenin signaling. As opposed to other ALL subtypes, E2A-PBX1 ALL cells express the β-catenin cofactors TCF3 and LEF1 at 〉5-fold higher levels and WNT16 at 〉12-fold higher levels compared to BCR-ABL1, MLL-AF4, Hyperdiploid and TEL-AML1-driven ALL subtypes. Conclusions: Constitutive activation of the canonical WNT/β-catenin signaling pathway in E2A-PBX1 ALL cells may explain the distinct role of pre-B cell receptor signaling in this ALL subset: Since pre-B cell receptor signaling via BTK negatively regulates WNT/β-catenin-dependent survival and self-renewal signaling (James et al., 2009), the level of constitutive WNT/β-catenin-signaling may determine permissiveness of ALL cells to pre-B cell receptor function. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3263 Background: Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), the development of drug resistance and long-term side effects of current treatments warrant new treatment modalities. Survivin/BIRC5, an inhibitor of apoptosis protein, is critical for the survival and proliferation of cancerous cells, is expressed in AML and ALL cells, and has been implicated in leukemia relapse. In the present study, we test the hypothesis that survivin is critical to the pathway of self-renewal of drug-resistant ALL cells. Methods: For gain of function studies, primary ALL cells were transduced with lentiviral survivin IRES GFP reporter (Survivin GFP) or empty GFP as a control. For loss of function studies, an inducible lentiviral shRNA vector expressing tRFP upon induction with doxycyclin was used. For in vitro evaluation of Survivin, CFU assays were used to monitor self-renewal capability, MTT assays and Trypan blue counts for viability determination were used for drug testing. For in vivo experiments, we used a NOD/SCID IL2Rγ−/- xenograft model with patient-derived ALL cells. Results: Survivin overexpression in primary ALL cells led in vitro to 4-fold more colonies than control in primary and secondary CFU assays and to increased resistance against Vincristine, Dexamethasone and L-Asparaginase (VDL) compared to controls (p

Description: Abstract 2167 Poster Board II-144 The transcriptional repressor BCL6 was discovered as a potent protooncogene in diffuse large B-cell lymphoma (DLBCL) and is also expressed in normal germinal center (GC) B cells. BCL6 protects DLBCL and GC B cells against DNA damage-induced apoptosis by suppressing target genes including p53, p21, ATR and p27. In preliminary experiments for this study, we found that treatment of BCR-ABL1-driven Ph+ ALL cells with Imatinib results in a 60- to 90-fold up-regulation of BCL6, reaching protein levels as high as in DLBCL and GC B cells. Since the oncogenic BCR-ABL1 kinase also drives leukemic transformation in CML cells, we studied regulation of BCL6 in human CML cells upon TKI-treatment. Like in Ph+ ALL, TKI-treatment resulted in rapid BCL6 mRNA and protein upregulation in CML cells, albeit to lesser degree than in Ph+ ALL and DLBCL. We hypothesized that upregulation of BCL6 in CML cells in response to TKI-treatment serves the same function as in DLBCL and protects from p53-mediated apoptosis. Through transcriptional repression of p53, p21, ATR and p27 as in DLBCL, upregulation of BCL6 in response to TKI-treatment would represent a defense mechanism of CML cells to evade TKI-induced cell death. We tested the role of BCL6 in CML in a genetic loss-of-function experiment: To this end, myeloid progenitor cells from bone marrow of BCL6+/+ and BCL6-/- mice were transformed with BCR-ABL1. Compared to their BCL6+/+ counterparts, BCL6-/- mouse CML cells were highly sensitive to Imatinib-treatment. In a complementary approach, we treated human CML cells with either Imatinib alone or with a novel BCL6 peptide inhibitor (Retro-inverso BCL6-peptide inhibitor, RI-BPI). Consistent with findings in BCL6-/- mouse CML cells, also human CML cells were significantly sensitized to Imatinib by BCL6 inhibition (using 5 mmol/l RI-BPI). Of note, the BCL6 inhibitory peptide RI-BPI alone induced no significant toxicity in human CML cells but synergized with Imatinib-treatment. Since RI-BPI alone had no cytotoxic effect on CML cells, we tested whether loss of BCL6 function affects self-renewal capacity of CML cells. This possibility was explored in two complementary colony formation assays: In one set of experiments, we tested the ability of BCL6+/+ and BCL6-/- mouse CML cells to form colonies in semisolid methylcellulose agar. The comparison between BCL6+/+ (94 ± 11 colonies) and BCL6-/- (