ALBERT

Beschreibung: Retroviral vector-mediated transduction of the BCR-ABL tyrosine kinase into Arf-deficient mouse bone marrow progenitors allows rapid ex vivo outgrowth of pure pre-B cell populations that induce a highly aggressive form of ALL when inoculated intravenously into healthy syngeneic mice. These leukemias resist therapy with the BCR-ABL kinase inhibitor imatinib, and surprisingly, drug resistance is non-tumor-cell autonomous. We now demonstrate that expression of the p185 BCR-ABL isoform (hereafter p185) in Arf-null pre-B cells is sufficient to generate polyclonal populations of leukemia-initiating cells (LICs), only 20 of which induce fatal, imatinib-resistant disease within one month after infusion. These p185+ Arf-null LICs are pre-B cells by immunophenotypic criteria, lack evidence of myeloid and stem cells markers, exhibit immunoglobulin heavy chain gene rearrangements and random proviral insertions, and are almost universally capable of initiating lethal, transplantable leukemias in healthy syngeneic recipient mice. Therefore, these LICs do not represent rare ‘cancer stem cells’. Resistance to targeted therapy with imatinib in this model system is not dependent upon mutations within the BCR-ABL kinase domain, but, rather, is mediated through interactions between the LICs and the host environment. While cytokines like IL-7 are dispensable for the efficient generation of p185+, Arf-null cells, these LICs remain IL-7-responsive and are significantly protected from imatinib-induced cytostasis by IL-7 in vitro. Introduction of p185 into Arf-null hematopoietic progenitors that also lack the common gamma chain (γc) for cytokine receptors bypasses their cytokine requirements and permits pre-B cell development. These p185+, Arf-null, γc-null LICs also initiate ALL but exhibit much greater sensitivity to imatinib in vivo, allowing a significant fraction of treated mice to sustain long term remissions even when therapy is discontinued. Therefore, salutary cytokines expressed in the hematopoietic microenvironment facilitate leukemic proliferation and confer resistance to targeted therapy. Not surprisingly, single agent therapy with dasatinib, a second generation BCR-ABL kinase inhibitor, is dramatically more efficacious in this pre-clinical Ph+ ALL model, although some recipient mice still succumb to disease despite continuous drug treatment. Using vectors co-expressing p185 and luciferase, we are now initiating dasatinib therapy when recipient animals have detectable disease burdens, thereby permitting a more dynamic assessment of therapeutic response and subsequent failure. We speculate that in human Ph+ ALL, the frequent deletion of the INK4A-ARF locus (∼50% of cases at diagnosis) might help to maintain LIC survival in the face of targeted therapy and so facilitate the emergence of drug-resistant BCR-ABL variants. As such, INK4A-ARF deletion might prove to be a poor prognostic indicator.

Beschreibung: The introduction of cultured p185BCR-ABL-expressing (p185+) Arf−/− pre-B cells into healthy syngeneic mice induces aggressive acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease. We adapted this high-throughput Philadelphia chromosome–positive (Ph+) ALL animal model for in vivo luminescent imaging to investigate disease progression, targeted therapeutic response, and ALL relapse in living mice. Mice bearing high leukemic burdens (simulating human Ph+ ALL at diagnosis) entered remission on maximally intensive, twice-daily dasatinib therapy, but invariably relapsed with disseminated and/or central nervous system disease. Although relapse was frequently accompanied by the eventual appearance of leukemic clones harboring BCR-ABL kinase domain (KD) mutations that confer drug resistance, their clonal emergence required prolonged dasatinib exposure. KD P-loop mutations predominated in mice receiving less intensive therapy, whereas high-dose treatment selected for T315I “gatekeeper” mutations resistant to all 3 Food and Drug Administration–approved BCR-ABL kinase inhibitors. The addition of dexamethasone and/or L-asparaginase to reduced-intensity dasatinib therapy improved long-term survival of the majority of mice that received all 3 drugs. Although non–tumor-cell–autonomous mechanisms can prevent full eradication of dasatinib-refractory ALL in this clinically relevant model, the emergence of resistance to BCR-ABL kinase inhibitors can be effectively circumvented by the addition of “conventional” chemotherapeutic agents with alternate antileukemic mechanisms of action.

Beschreibung: Abstract 540 Expression of the constitutively active tyrosine kinase BCR-ABL1 is the hallmark of two diseases with distinct pathologic and clinical features: chronic myeloid leukemia (CML), an expansion of relatively mature granulocytes that typically responds well to kinase inhibition, and pre-B cell acute lymphoblastic leukemia (ALL), an aggressive malignancy of lymphoid progenitors that has a dismal prognosis. The basis for this dichotomy has been poorly understood. Recent studies profiling genome-wide DNA copy number alterations in CML and ALL have identified common deletions of IKZF1 (encoding the lymphoid transcription factor IKAROS) in de novo BCR-ABL1 positive ALL, and at the progression of CML to lymphoid blast crisis, suggesting that perturbation of IKAROS activity is a key event in the pathogenesis of BCR-ABL1 lymphoid leukemia. The IKAROS alterations commonly involve coding exons 3–6, resulting in expression of a dominant negative IKAROS isoform, IK6. Moreover, the presence of IKZF1 alterations is associated with poor outcome in BCR-ABL1 ALL. We have previously shown in a retroviral bone marrow transplant model of BCR-ABL1 ALL that Ikzf1 loss results in increased penetrance of leukemia, but the role of IK6 in the pathogenesis of ALL has not been studied. Here, we have examined the effect of the expression of Ik6 in a retroviral bone marrow transplant model of murine BCR-ABL1 B-progenitor ALL. Unmanipulated marrow from C57BL/6 Arf null mice was transduced with MSCV retrovirus coexpressing p185 BCR-ABL1 and luciferase, plated for 8 days to derive pre-B cells, then transduced with MSCV retrovirus expressing either wildtype Ikaros (Ik1-RFP), Ik6-RFP, or empty vector. Expression of Ik1 was not tolerated and resulted in cell death and apoptosis. IK6 expression led to increased proliferation of p185+Arf null cells with reduced sensitivity to the BCR-ABL1 kinase inhibitor dasatinib compared to cells transduced with empty vector. Intracellular phosphosignaling analysis of Crkl phosphorylation demonstrated that this reduced sensitivity to dasatinib was independent of ABL1 inhibition. Gene expression profiling of p185+Arf null-Ik6 cells revealed a gene expression signature similar to that of human BCR-ABL1+ ALL with enrichment of hematopoietic stem cells genes as well as genes involved in B-cell receptor, Notch, and Jak-Stat signaling pathways. To test the role of Ik6 in leukemogenesis and treatment responsiveness in vivo, p185 BCR-ABL1-luciferase Arf null cells were transduced with MSCV retrovirus expressing GFP alone, Ik1-GFP, or Ik6-GFP then transplanted into lethally irradiated C57BL/6 recipients. Expression of Ik6 in vivo led to accelerated tumorigenesis and decreased survival with tumors uniformly of pre-B immunophenotype. Moreover, mice transplanted with Ik6-expressing marrow were less sensitive to dasatinib therapy (10mg/kg QD initiated 7 days post-BMT) compared to control mice (19d vs. 31.5d, p

Beschreibung: Although sickle cell disease (SCD) is a monogenic disorder, the severity and specific organ dysfunction and failure are strongly influenced by genetic modifiers. Rapid identification of all modifiers in patients and well-phenotyped cohorts will better define the impact of relevant variants on clinical status, inform disease biology, and identify new therapeutic strategies. We created the Sickle Genome Project (SGP), a whole genome sequencing (WGS) strategy, to define genomic variation and modifiers of SCD. We performed WGS on 871 African American SCD patients from St. Jude Children's Research Hospital who participated in the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer, 2018) and Texas Children's Hospital Hematology Center (TCHC). We developed robust pipelines for accurate detection of single nucleotide polymorphisms (SNPs), identification of structural variants and data retrieval/sharing via the St. Jude Cloud platform (to be described elsewhere). Notable findings include: 1) Confirmed associations of common genetic modifiers with SCD phenotypes, including levels of fetal hemoglobin (BCL11A, HBS1L-MYB, HBB), bilirubin (UGT1A1), and microalbuminuria (APOL1). Additional associations approaching genome-wide significance require further investigation, including replication in independent samples. 2) Improved determination of the SCD modifier α-thalassemia. The most common α-thalassemia mutations in SCD are 3.7 kb or 4.2 kb deletions (-α3.7 and -α4.2 alleles), which arose from recombination between homologous HBA1 and HBA2 genes and are difficult to map using standard WGS reads. Three independent crossover events are described for -α3.7 and one for -α4.2 in SCD cohorts. We developed a novel approach to identify α-globin gene deletions by local de novo assembly of WGS data and coverage depth analysis. We identified 5 -α3.7 alleles (frequencies 0.77-32.12%) and 7 -α4.2 alleles (frequencies 0.19-5.77%). Collectively, the frequency of all -α alleles was 57%, reflecting at least 12 distinct recombination events, greatly exceeding previously published counts. These findings better define the evolution of α-globin genes to allow improved understanding of their regulation and influence on SCD. 3) Characterization of β0-thalassemia alleles. Mutations in the extended β-globin locus influence SCD phenotypes. Five SGP patients had large β-globin (HBB) deletions associated with elevated fetal hemoglobin, which ameliorates symptoms of SCD. Twenty-three patients had HbSβ0-thalassemia, which reduces the severity of some SCD phenotypes. Overall, 48.6% (18/37) of patients clinically designated as HbSβ0 -thalassemia had no identified β-thalassemia mutation. Moreover, 4/680 patients (0.6%) designated HbSS were identified to be β0-thalassemia heterozygotes. The MCV, RBC and %HbA2 distributions overlapped substantially in correct vs. incorrect genotype assignments. Improved discrimination of HbSβ0 vs HbSS genotypes by WGS will better define associated phenotype differences to impact clinical care. 4) Determination of a genetic variant linked to vaso-occlusive crisis (VOC). Previously, a single GWAS study linked rs3115229, located 63.7 kb 5′ upstream of the KIAA1109 gene, with VOC at borderline significance (P = 5.63 × 10−8) (Chaturvedi et al, Blood 130, 2017). Using WGS data for 327 SGP participants (HbSS or HbSβ0-thalassemia) enrolled in the SCCRIPP study, we found strong association (p = 7 x 10-5) between the onset of VOC and a 4-SNP diplotype within an adjacent LD block of the KIAA1109-TENR-IL2-IL21 region (chr4: 122.8Mb - 123.8Mb) which has been previously associated with numerous inflammatory disorders. We validated this association using imputed genome-wide array data in an independent group of SCD patients (Sleep and Asthma Cohort, n= 181 patients, p = 0.05) (Cohen et al, Ann Am Thorac Soc, 2016). This works provides confirmation that the region surrounding KIAA1109 is associated with pain crisis in SCD. Our studies provide new information on the genomic architecture of SCD patients and delineate a consolidated approach for future applications of precision medicine. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy. Estepp:Global Blood Therapeutics: Consultancy, Research Funding; ASH Scholar: Research Funding; NHLBI: Research Funding; Daiichi Sankyo: Consultancy.

Beschreibung: Despite the dramatic efficacy of imatinib and second-generation BCR-ABL kinase inhibitors (dasatinib and nilotinib) in chronic myelogenous leukemia (CML), these drugs fail to provide durable therapeutic benefit in patients with Philadelphia-chromosome positive (Ph+) BCR-ABL-induced acute lymphoblastic leukemias (ALL). In contrast to chronic-phase CML, deletion of the CDKN2A (INK4A-ARF) tumor suppressor locus occurs in diagnostic blast samples from 65% of Ph+ ALL patients. Introduction of cultured Arf−/− p185BCR-ABL-expressing (p185+) pre-B cells, but not their Arf+/+ counterparts, into healthy syngeneic recipient mice induces fulminant B cell leukemia. Virtually every p185+, Arf−/− cell has leukemia-initiating cell capacity. We have now monitored disease progression and dasatinib-responsiveness in vivo by following the fate of leukemogenic p185+ Arf−/− donor cells engineered to express luciferase. When dasatinib therapy is initiated in recipients with low leukemic burdens, most animals remain in remission for many weeks, but some ultimately relapse on therapy or soon after drug discontinuation. Most resistant leukemias sustained kinase domain (KD) mutations, most frequently in the ATP-binding (‘P’) loop, that only modestly impact drug sensitivity. In contrast, in animals bearing high leukemic burdens (simulating the human clinical condition at diagnosis), dasatinib therapy induced dramatic reductions in luminescent signals, but all animals harbored persistent, measurable deposits of drug-resistant cells. Residual drug-refractory ALL cells recovered from these otherwise healthy animals contained rare clones expressing the T315I mutation known to confer drug resistance to imatinib, dasatinib and nilotinib, and most frequently associated with clinical resistance in Ph+ ALL. However, the vast majority of BCR-ABL alleles were mutation-free, implying that KD mutations could not be solely responsible for drug resistance. Following continued therapy, these mice underwent clinical relapse preceded by dramatic increases in luminescent signals, both in the hematopoietic compartment and central nervous system. Many of these drug-resistant leukemias now harbored the T315I KD mutation. Animals that had been maintained in remission with 4 weeks of continuous dasatinib therapy quickly relapsed upon therapy discontinuation, almost always without evidence of BCR-ABL KD mutations. Together, these studies indicate that in this clinically-relevant Ph+ ALL model, several factors including Arf lossof- function, disease burden, intensity of therapy, and length of drug exposure interact to determine therapeutic outcome and to trigger confluent mechanisms of drug resistance. Critically, while continuous dasatinib therapy efficiently selects for and maintains cells harboring drug-resistant KD mutations that mediate eventual clinical relapse, most drugrefractory leukemic cells survive in the absence of KD mutations during maintenance therapy, implying that mutation-independent factors facilitate persistence of residual leukemic disease. We propose that Arf inactivation in Ph+ ALL (but not in CML) enhances the biological ‘fitness’ of leukemic cells and diminishes the efficiency with which targeted therapy can successfully eradicate drug-refractory disease. This facilitates the subsequent emergence of cell-intrinsic drug resistance, most frequently manifested as BCR-ABL KD mutations.