ALBERT

Description: Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNA-like ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN.

Description: Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Most cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. Herein, we report the preliminary description of a novel transgenic mouse line that expresses G192X Ela2, reproducing the G193X ELA2 mutation found in some patients with SCN. The G192X mutation was introduced into the murine Ela2 locus by homologous recombination in embryonic stem cells. Heterozygous or homozygous G192 Ela2 “knock-in” mice were healthy with no apparent developmental defect. While expression of Ela2 mRNA was normal, no mature NE protein was detected in the neutrophils of homozygous G192X Ela2 mice. However, in granulocytic precursors (mainly promyelocytes/myelocytes) a small amount of heavily glycosylated mutant NE protein was detected. Together, these observations suggest that G192X NE protein is retained in the endoplasmic reticulum (ER) and rapidly degraded. Consistent with ER stress and induction of the UPR, a significant increase in BiP/GRP78 and ATF6 mRNA expression in mutant granulocytic precursors were observed. Surprisingly, G192X Ela2 mice have normal basal granulopoiesis. The number of circulating neutrophils, granulocytic differentiation in the bone marrow, and number and cytokine responsiveness of myeloid progenitors were comparable to wild type mice. In summary, the G192X Ela2 mice appear to reproduce the NE protein misfolding and UPR activation observed in human SCN granulocytic precursors. However, expression of G192X Ela2 is not sufficient to disrupt basal granulopoiesis in mice. Studies of stress granulopoiesis are underway.

Description: Introduction The BCR-ABL1 gene fusion is the most common molecular aberration in adult ALL and confers an adverse prognosis. The incorporation of ABL1 tyrosine kinase inhibitors (TKIs) into ALL chemotherapy regimens has dramatically improved outcomes for BCR-ABL1+ ALL. Subsequently, reduced-intensity TKI-based regimens have been shown to be as effective and less toxic than intensive TKI-based regimens (Foa et al. Blood 2011; Chalandon et al. Blood 2015). Asciminib (ABL001) is a potent, specific allosteric inhibitor of ABL1 that binds to a site spatially distinct from the catalytic domain. Asciminib was developed for efficacy against BCR-ABL1 mutations conferring resistance to standard TKIs. Preclinical studies demonstrated that the combination of asciminib and TKI leads to sustained disease control in a cell line xenograft model of BCR-ABL1+ CML (Wylie et al. Nature 2017). We also observed that the addition of asciminib to TKI significantly prolongs survival in patient-derived xenograft models of BCR-AB1L+ ALL, even in the context of a pre-existing subclonal T315I mutation (unpublished). An ongoing phase I Novartis study (NCT02081378) has shown safety and efficacy of asciminib alone and in combination with TKIs in relapsed/refractory (R/R) BCR-ABL1+ ALL and CML. We hypothesized that dual ABL blockade with catalytic domain and allosteric inhibitors would be tolerable and effective in older adults with BCR-ABL1+ ALL. Methods This is an investigator initiated, phase I study (NCT03595917) of asciminib in combination with dasatinib plus prednisone in BCR-ABL1+ ALL. The study uses a 3+3 dose escalation design with an expansion cohort with the primary objective to determine the MTD or RP2D. Secondary objectives include defining the depth of molecular remissions at early time points and durability of responses. Patients (pts) ≥ 50 years (yrs) with newly diagnosed BCR-ABL+ ALL or CML in blast phase with p190 or p210 transcripts are eligible. Younger pts (18-49 yrs) unfit for intensive chemotherapy, or pts ≥ 18 yrs with ALL R/R to intensive chemotherapy are also eligible (excluded if prior dasatinib or asciminib exposure). Pts are treated with dasatinib 140 mg daily and prednisone 60 mg/m2 days 1-24 (max 120 mg daily, tapered days 25-32) following Foa et al with escalating doses of asciminib once daily administered fasting (DL1: 40 mg; DL2: 80 mg; DL3: 160 mg; DL: 320 mg). Pts receive CNS prophylaxis with intra-thecal chemotherapy. Transplant-eligible pts are recommended to be consolidated with allogeneic transplant as medically appropriate at or after day 85; transplant-ineligible pts may remain on therapy if deriving clinical benefit. Correlative science efforts aim to identify biomarkers of response to combined BCR-ABL1 blockade and molecular signatures enriched in minimal residual disease, based on targeted tumor sequencing and serial single-cell RNA sequencing of leukemia and stromal populations from blood and marrow. Results As of July 31, 2019, 6 adults (3 male/3 female) have enrolled and 3 have completed DL1. All 6 had newly diagnosed, untreated BCR-ABL1+ ALL. The median age at registration was 67 (range 53 - 83) yrs. Three pts treated on DL1 have been evaluated for DLT and discontinued therapy (1 per pt decision after 4 cycles; 2 pts bridged to transplant after 4 cycles). At the time of this report, there has been no induction mortality, no DLTs, and no grade 3 or higher AEs at DL1. At DL1, 1 pt was treated entirely outpatient, the other 2 pts required short hospital stays (4 and 5 days) after treatment initiation. Responses for the 3 pts treated at DL1 are summarized in the table. Pre-treatment tumor genotyping and serial on-treatment, single-cell profiling of tumor and microenvironment fractions from marrow and blood have been performed in all pts to date and have revealed biophysical, transcriptional, and genetic inter-tumor heterogeneity. These data will be used to illuminate disease biology and define predictive biomarkers, pending further maturation of clinical endpoints. Conclusion Dual ABL1 kinase inhibition with asciminib and dasatinib plus prednisone in newly diagnosed BCR-ABL1+ ALL is feasible in older adults with no increase in toxicity. The last pt at DL2 is completing treatment and being evaluated for DLT before planned escalation to DL3. An expansion cohort of 10 pts at the MTD or RP2D is planned to further characterize safety and clinical activity of the combination. Disclosures Stevenson: Celgene: Research Funding. Weinstock:Celgene: Research Funding. DeAngelo:Incyte: Consultancy; Pfizer: Consultancy; Jazz Pharmaceuticals Inc: Consultancy; Novartis: Consultancy, Research Funding; GlycoMimetics: Research Funding; Abbvie: Research Funding; Shire: Consultancy; Celgene: Consultancy; Takeda Pharmaceuticals: Consultancy; Amgen: Consultancy; Blueprint: Consultancy, Research Funding.

Description: Lymphomas represent nearly 70 distinct diseases with unique clinical presentations, therapeutic responses and underlying biology. There is a pressing shortage of publically available cell line and in vivo models of nearly all of these diseases, which has severely hampered efforts to understand and target their biology. To address this issue, we have established a repository of patient-derived xenografts (PDX) of lymphomas by engrafting human tumors into immunodeficient NOD/SCID/IL2rgnull (NSG) mice. These lymphomas, along with a spectrum of other PDXs of hematologic malignancies, are available to collaborators through the online portal PRoXe (Public Repository of Xenografts) at http://PRoXe.org. Blood and bone marrow specimens involved with tumor are injected by tail vein (IV) injection. Lymph node and extranodal biopsy specimens are implanted under the renal capsule as a 1x1x2mm tumor seed (renal), which maintains the in situ microarchitecture. A full description of xenografted lymphomas is included in the Table. Table 1.DiseaseType of implant# in 1st passage# in 2nd passage or higherT-cell prolymphocytic leukemiaIV1Angioimmunoblastic T-cell lymphomaIV11Mantle cell lymphomaIV12Double-hit DLBCLIV2Sézary SyndromeIV1Adult T-cell Leukemia/LymphomaIV1Diffuse large B cell lymphomaIV2Diffuse large B cell lymphomarenal2Marginal zone lymphomarenal11NK/T-cell lymphomarenal1Peripheral T-cell lymphoma-NOSrenal1Breast implant-associated anaplastic large cell lymphomarenal1 Engrafted PDXs have been extensively characterized by immunohistochemistry, flow cytometry, transcriptome sequencing and targeted DNA sequencing. Flow cytometric analysis of patient tumors and their respective xenografts consistently revealed highly concordant immunophenotypes compared to the original tumors. Similarly, immunohistochemistry of involved tissues confirmed retention of tumor immunophenotypes, architecture, and even tissue tropism in the PDXs. Examples include a Sézary syndrome PDX that was injected by tail vein and trafficked to spleen, bone marrow, blood and skin, a diffuse large B-cell lymphoma (DLBCL) PDX that infiltrated the CNS, and a second DLBCL PDX that was implanted into the renal capsule of the left kidney and progressed within 8 weeks to bilateral renal involvement. Other notable models include a breast implant-associated, ALK-negative anaplastic large cell lymphoma implanted under the renal capsule that metastasized to the liver and spleen while uniformly retaining CD30 positivity. Two double-hit lymphoma (DHL) PDXs maintained their CD20-negative phenotype through serial passage to P1. A peripheral T-cell lymphoma-NOS (PTCL) specimen implanted under the renal capsule engrafted in the spleen, with a notable admixture of nonmalignant T cells and scattered EBV-positive B cells. T-cell receptor gene rearrangement PCR performed on this PTCL demonstrated an identical rearrangement pattern in the primary tumor and the PDX. Luciferized mantle cell lymphoma and DHL PDXs clearly home to bone marrow, lymph nodes, spleen, and liver as early as two weeks after injection. These findings support the utility of these PDX lines as in vivo models that more accurately recapitulate the human disease than commonly used subcutaneous cell line models. In addition to generating PDXs that remain faithful to their source tumors, we have witnessed interesting examples of in vivo histologic transformation, opening the door to studies of disease progression. One primary follicular lymphoma specimen injected into a cohort of mice transformed to DLBCL in one mouse and a lymphoblastic lymphoma-like disease in another mouse, as confirmed by IHC and flow cytometry. Further xenografting of primary tumors is underway with the goal of establishing a large repository of lymphoma PDXs useful for biologic interrogation and preclinical trials. Disclosures Davids: Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Shipp:Gilead: Consultancy; Sanofi: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees.

Description: To expedite the translation of biologic discoveries into novel therapeutics, there is a pressing need for panels of in vivo models that capture the molecular complexity of human disease. While traditional cell lines and genetically engineered mouse models are useful tools, they are insufficient to assess the broad diversity of human tumors within a context that recapitulates in situ biology. Patient-derived xenografts (PDXs), generated by transplanting primary human tumor cells into immune-deficient NOD.Cg-Prkdcscid/Il2rgtm1Wjl/SzJ (NSG) mice, surmount some of the limitations of these traditional platforms and have been increasingly utilized as tools for preclinical investigation. However, the infrastructure required to generate, bank, and characterize PDX models limits their availability to only a few investigators. To address this issue, we established a repository of PDX models of leukemia and lymphoma, which we have named the Public Repository of Xenografts (PRoXe). At the time of this writing, PRoXe contains 213 independent lines that have been passaged through mice once (P0), 123 of which have been repassaged in a second generation (P1) or further repassaged. The repository encompasses AML, B- and T-ALL, and B- and T-cell non-Hodgkin lymphoma (NHL) across a range of cytogenetic- and molecularly-defined subtypes (Table 1). PRoXe is extensively annotated with patient-level information, including demographics, phase of treatment, prior therapies, tumor immunophenotye, cytogenetics, and molecular diagnostics. PDX lines available for distribution are characterized by immunophenotyping, whole transcriptome sequencing (RNAseq), and targeted exon sequencing of ~300 genes. To confirm fidelity of engrafted tumors to their corresponding clinical samples, lymphomas were morphologically assessed in P0 mice by H&E and, when pathologic adjudication was required, by immunohistochemistry. Xenografted leukemias were compared to their original tumors immunophenotypically. Unsupervised hierarchical clustering was performed on 132 of these lines based on transcriptome sequencing data and demonstrated 94% concordance between classification of the PDX lines by RNA expression and by the annotated clinical-pathologic diagnoses. Discordant cases highlighted unusual variants, such as B-ALL with aberrant expression of myeloid markers and a follicular lymphoma that underwent blastic transformation in the mouse. Multiple lines have been luciferized and confirmed to home to bone marrow, spleen, and liver. Existing lines from PRoXe have already been shared with more than ten academic laboratories and multiple industrial partners. All of the data referenced here are freely available through a customized web-based search application at http://proxe.org, and lines can be requested for in vitro or in vivo experiments. We are actively expanding the size of PRoXe to allow for large pre-clinical studies that are powered to detect differences across genetically defined subsets. Thus, we are happy to host additional lines from outside investigators on PRoXe and thereby expand the availability of these valuable reagents. Finally, we have made the source code for PRoXe (in R Shiny) open-access, so that other investigators can establish their own portals. Table 1. WHO diagnostic entities encompassed within PRoXe at P1 or later, or P0 or later for B-ALLs. WHO Classification - number of lines per diagnostic entity AML, Other Myeloid, and Ambiguous Lineage [n=32] ALL [n=107] AML - recurrent gene mutations 6 B-ALL - NOS 44 AML - MDS-related changes 5 B-ALL - MLL-rearranged 11 AML - NOS 4 B-ALL - BCR-ABL 10 AML - MLLT3-MLL 2 B-ALL - hyperdiploidy 9 Acute myelomonocytic leukemia 1 B-ALL - TEL-AML1 8 Acute monocytic leukemia 1 B-ALL - E2A-PBX1 3 AML unable to classify 2 B-ALL unable to classify 1 Blastic plasmacytoid dendritic cell neoplasm 8 T-ALL 21 Mixed phenotype, MLL rearranged 1 B/myeloid acute leukemia 1 Myelodysplastic syndrome 1 Mature B cell neoplasms[n=11] Mature T and NK cell neoplasms [n=4] DBLCL - NOS 4 Angioimmunoblastic T-cell lymphoma 1 Mantle cell lymphoma 3 Adult T-cell leukemia/lymphoma 1 Extranodal marginal zone lymphoma 1 Extranodal NK/T-cell lymphoma 1 B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL 3 SŽzary syndrome 1 Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Etchin:Karyopharm: Research Funding. Lane:Stemline Therapeutics, Inc.: Research Funding. Stone:Abbvie: Consultancy; Novartis: Research Funding; Celator: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Merck: Consultancy; Karyopharm: Consultancy; Roche/Genetech: Consultancy; Pfizer: Consultancy; AROG: Consultancy; Juno: Consultancy.

Description: In order to better understand the pathogenesis of acute promyelocytic leukemia (APL, FAB M3), we sought to determine its gene expression signature by comparing the expression profiles of 14 APL samples to that of other AML subtypes (M0, M1, M2, M4, n=62) and to fractionated normal whole bone marrow cells (CD34 cells, promyelocytes, PMNs, n=5 each). We used ANOVA and SAM (Significance Analysis of Microarrays) to select genes that were highly expressed in APL cells and that displayed low to no expression in other AML subtypes. The APL signature was then further refined by filtering genes whose expression in APL was not significantly different from that of normal promyelocytes, yielding 1121 annotated genes that reliably distinguish APL from the other FAB subtypes using unsupervised hierarchical clustering, both in training and validation datasets. Fold change differences in expression between M3 and other AML FAB classes were striking, for example: GABRE 35.4, HGF 21.3, ANXA8 21.3, PTPRG 16.9, PTGDS 12.1, PPARG 11.1, STAB1 9.8. A large proportion of the APL versus other FAB dysregulome was recapitulated when we compared APL expression to that of the normal pattern of myeloid development. We identified 733 annotated genes with significantly different expression in APL versus normal myeloid cell fractions. These dysregulated genes were assigned to 4 classes: persistently expressed CD34 cell-specific genes, repressed promyelocyte-specific genes, prematurely expressed neutrophil-specific genes and genes with high expression in APL and low/no expression in normal myeloid cell fractions. Expression differences in several of the most dysregulated genes were validated by qRT-PCR. We then examined the expression of the APL signature genes in myeloid cell lines and tumors from a murine APL model. The bona fide M3 signature was not apparent in resting NB4 cells (which contain t(15;17), and which express PML-RARA), nor in PR-9 cells following Zn induction of PML-RARA expression, suggesting that neither cell line accurately models the gene expression signature of primary APL cells. Most of the nodal genes of the mCG-PML-RARA murine APL dysregulome (Yuan, et al, 2007) are similarly dysregulated in human M3 cells; however, the human and mouse dysregulomes do not completely coincide. Finally, we have begun investigating which APL signature genes are direct transcriptional targets of PML-RARA. The promoters of the APL signature genes were analyzed for the presence of known PML-RARA binding sites using multiple computational methods. The analyses demonstrated that several transcription factors (EBF3, TWIST1, SIX3, PPARG) have putative retinoic acid response elements (RAREs) in their upstream regulatory regions. Additionally, we examined the promoters of some of the most upregulated genes (HGF, PTGDS, STAB1) for known consensus sites of these transcription factors, and found that all have putative binding sites for at least one. These results suggest that PML-RARA may initiate a transcriptional cascade that relies not only on its own activity, but also on the actions of downstream transcription factors. In summary, our studies indicate that primary APL cells have a gene expression signature that is consistent and highly reproducible, but different from commonly used human APL cell lines and a mouse model of APL. The molecular mechanisms that govern this unique signature are currently under investigation.