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Supplemental Figures 1-3, Tables 1-3. Supplemental Figure 1. Highly variable expression of MMRN1 in pediatric AML patient specimens. Supplemental Figure 2. Clinical outcome in patients with high and low MMRN1 expression. Supplemental Figure 3. Relationship between bone marrow blast percentage and MMRN1 expression in the AAML0531 patient cohort. SUPPLEMENTAL TABLE 1. Comparison of Baseline Characteristics of Patients with Low (

Purpose: Exploratory gene expression array analyses suggested multimerin-1 (MMRN1) to be a predictive biomarker in acute myelogenous leukemia (AML). Following up on these studies, we evaluated the role of MMRN1 expression as outcome predictor in two recent Children's Oncology Group trials.Experimental Design: We retrospectively quantified MMRN1 expression in 183 participants of AAML03P1 and 750 participants of AAML0531 by reverse-transcriptase PCR and correlated expression levels with disease characteristics and clinical outcome.Results: In AAML03P1, the highest quartile of MMRN1 expression (expression ≥0.5 relative to β-glucuronidase; n = 45) was associated with inferior event-free survival (EFS; P < 0.002) and higher relapse risk (P < 0.004). In AAML0531, in which we quantified MMRN1 mRNA for validation, patients with relative MMRN1 expression ≥0.5 (n = 160) less likely achieved remission (67% vs. 77%, P = 0.006), and more frequently had minimal residual disease (43% vs. 24%, P = 0.001) after one induction course. They had inferior overall survival (OS; 44% ± 9% vs. 69% ± 4% at 5 years; P < 0.001) and EFS (32% ± 8% vs. 54% ± 4% at 5 years; P < 0.001) and higher relapse risk (57% ± 10% vs. 35% ± 5% at 5 years; P < 0.001). These differences were partly attributable to the fact that patients with high MMRN1 expression less likely had cytogenetic/molecular low-risk disease (P < 0.001) than those with low MMRN1 expression. Nevertheless, after multivariable adjustment, high MMRN1 expression remained statistically significantly associated with shorter OS (HR, 1.57; 95% confidence interval, 1.17–2.12; P = 0.003) and EFS (HR, 1.34; 1.04–1.73; P = 0.025), and higher relapse risk (HR, 1.40; 1.01–1.94; P = 0.044).Conclusions: Together, our studies identify MMRN1 expression as a novel biomarker that may refine AML risk stratification. Clin Cancer Res; 21(14); 3187–95. ©2015 AACR.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesisin vivo, thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs.In vitro, dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.Graphical abstract

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal targeting epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33V-set/CD3 bispecific antibody (BsAb) and CD33V-set-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33PAN antibodies”). These antibodies internalized when bound to CD33 and, as CD33PAN/CD3 BsAb, had potent cytolytic effects against CD33+ cells. Together, our data provide rationale for further development of CD33PAN antibody-based therapeutics.

Over 26,000 people will face a new diagnosis of acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) in 2020 in the U.S. alone, and only a minority will be long-term survivors [1]. Am...

Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia and higher-risk myelodysplastic syndrome (MDS). Of increasing interest are alpha-particle-emitting radionuclides such as astatine-211 (

Background:Improved survival with gemtuzumab ozogamicin (GO) in some people with acute myeloid leukemia has validated CD33 as immunotherapeutic target and sparked interested in developing new, highly potent CD33-directed therapeutics. As a limitation of this treatment strategy, CD33 expression on maturing and mature myeloid cells causes significant on-target, off-leukemia effects. Toxicity of CD33-targeted immunotherapy should be minimal in the presence of normal hematopoietic stem and progenitor cells (HSPCs) engineered to lack CD33 variants recognized by therapeutic antibodies. Indeed, very recent studies have shown that CRISPR/Cas9 with a single guide RNA (gRNA) designed to target the CD33coding region reduces display of CD33 and protects engineered cells from CD33 CAR T-cells. However, this approach resulted in low levels of CD33disruption in vivoand off-target activity. We therefore developed an approach in which the Cas9 protein is complexed with two synthetic gRNAs for precise excision of the intervening sequence (i.e. more controlled genome editing than what can be accomplished with a single gRNA). We directed these two gRNAs to intronic sequences for precise excision of exon 2, which encodes the V-set domain of CD33 that is recognized by all current CD33 therapeutics including GO. This approach eliminates exonic indels and protects engineered cells from CD33-targeted immunotherapy while maintaining expression of an exon 2-free variant of CD33 (CD33∆E2), which has previously been identified as natural isoform in human HSPCs. Methods: We used human myeloid ML-1 cells and human fetal liver CD34+ HSPCs for CRISPR/Cas9 editing, which was carried out by electroporation of purified Cas9 protein complexed with synthetic gRNAs. In vitro cytotoxicity assays were performed to test sensitivity to GO and the CD33/CD3 bispecific antibody AMG 330.For in vivoassessment of engineered HSPCs, NSG neonate mice were infused with 6.0x105human CD34+ cells, and peripheral blood analyzed biweekly for 14 weeks. Results:Delivery of Cas9/sgRNA ribonucleoproteins (RNPs) in ML-1 cells resulted in exon 2 deletion and time-dependent reduction in cell surface display of full-length CD33 (CD33FL). Pure populations of CD33∆E2cells were generated via single-cell cloning. These sublines lacked surface display of CD33FLbut, instead, expressed increased levels of the CD33∆E2transcript. CD33∆E2ML-1 cells were completely resistant to GO and AMG 330, whereas wild-type ML-1 cells were highly sensitive to these two drugs. Consistent with the findings in ML-1 cells, delivery of CRISPR/Cas9 RNPs to human fetal liver CD34+ HSPCs resulted in the expected CD33exon 2 deletion and decreased CD33FLsurface expression, while it did not impact the distribution of colony-forming cellsand only minimally reduced colony-forming potential. In NSG mouse xenotransplantation experiments, we found these CD33∆E2human CD34+ HSPCs to have comparable engraftment and multilineage differentiation potential relative to HSPCs expressing CD33FL(Fig. 1A). As expected, CD33FLexpression was substantially reduced in peripheral blood monocytes from CD33∆E2 HSPC-engrafted animals (Fig. 1B). In addition, robust engraftment of CD33∆E2 -engineered HSPCs in bone marrow was demonstrated by over 50% biallelic CD33exon 2 deletion at time of necropsy from colony-forming cells analysis (Fig. 1C). To determine if in vivodifferentiated myeloid CD33∆E2cells derived from infused human CD34+ HSPCs are indeed resistant to CD33-directed therapies, we treated mice with GO intravenously. GO administration reduced the number of circulating CD33FLCD14+ myeloid cells while leaving the number of CD33∆E2cells unaffected. Conclusion:These findings support a novel strategy in which CD33∆E2-engineeredHSPCs are used to widen the therapeutic window of CD33-directed immunotherapies. Such cells could be envisioned for people at risk for the need of CD33-immunotherapy, e.g. AML patients in remission (where CD33-immunotherapy could be used to prevent/treat relapse) or, pre-emptively, for people with genetic AML-predisposition syndromes (such that CD33-targeted immunotherapy could be given safely if AML occurred). With this potential, further development of CD33∆E2-engineered HSPCs toward clinical application is warranted. Disclosures Walter: Amphivena Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Aptevo Therapeutics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding; Covagen AG: Consultancy, Other: Clinical Trial Support, Research Funding; Actinium Pharmaceuticals, Inc: Other: Clinical Trial support , Research Funding; Amgen Inc: Other: Clinical Trial Support, Research Funding; Boehringer Ingelheim Pharma GmbH & Co. KG: Consultancy; Pfizer, Inc: Consultancy; Seattle Genetics, Inc: Consultancy, Other: Clinical Trial Support, Research Funding. Kiem:Magenta: Consultancy; Homology Medicine: Consultancy; Rocket Pharmaceuticals: Consultancy.

Background: Radioimmunotherapy (RIT) has long been pursued to improve outcomes in acute leukemia. Of current interest are alpha-particle emitting radionuclides as they deliver a very large amount of radiation over just a few cell diameters, enabling efficient and selective target cell kill. So far, alpha-emitters including astatine-211 (211At) have been primarily explored with monoclonal antibodies (mAbs) targeting CD45 or CD33 but their broad display on non-malignant target-expressing cells can lead to marked "on-target, off tumor cell" toxicities. To overcome this limitation, we developed a novel form of 211At-based RIT targeting CD123. CD123 is displayed widely on acute leukemia cells, including underlying leukemic stem cells, but is expressed only on a discrete subset of normal hematopoietic cells and is virtually absent on non-blood cells. Methods: We immunized BALB/c mice with peptides consisting of the extracellular domain of human CD123 to generate anti-CD123 mAbs. Flow cytometry-based assays with human acute leukemia cell lines were used to characterize binding of hybridoma supernatants and mAbs to CD123. mAbs were conjugated with isothiocyantophenethyl-ureido-closo-decaborate(2-) (B10), a boron cage molecule for subsequent astatination, and were then labeled with 211At. In vivo leukemia cell targeting ("biodistribution") and efficacy studies were conducted in immunodeficient NOD-Rag1 null IL2rɣ null/J (NRG) mice xenografted with MOLM-13 cells, a CD123+ human acute myeloid leukemia cell line. Results: Based on initial hybridoma screening studies, we selected 4 mAbs (10C4, 5G4, 11F11, and 1H8) for further characterization. Phenotyping studies with CD123+ and CD123- human acute leukemia cell lines (including CD123+ cell lines in which CD123 was deleted via CRISPR/Cas9) confirmed specific binding of all mAbs to human CD123 (binding intensity: 10C4>5G4=11F11=1H8), with 10C4 yielding a higher median fluorescence intensity than the widely used commercial anti-CD123 mAb clones, 7G3 and 6H6 (Figure 1). In vitro internalization with a panel of human acute leukemia cell lines studies demonstrated uptake of all mAbs by CD123+ target cells with a kinetic slower than that for anti-CD33 antibodies (typically, 30-50% of the anti-CD123 mAb internalized over 2-4 hours). All 4 anti-CD123 mAbs could be conjugated to B10 and subsequently labeled with 211At. Unlike a non-binding 211At-labeled control mAb, 211At-labeled anti-CD123 mAbs showed uptake at MOLM-13 flank tumors in NRG mice carrying MOLM-13 xenografts. After additional leukemia cell targeting studies to optimize the dosing of 10C4, we conducted proof-of-concept efficacy studies in NRG mice injected intravenously with luciferase-transduced MOLM-13 cells (disseminated leukemia model). Animals were either untreated or treated with 10 µCi, 20 µCi, or 40 µCi of 211At-labeled 10C4-B10 mAb (9-11 animals/group). This was followed by the infusion of bone marrow cells from donor mice as stem cell support 3 days later. As shown in Figure 2 and Figure 3, 211At-10C4-B10 led to a dose dependent decrease in tumor burden. Further, the treatment significantly prolonged survival compared to untreated animals (median survival: 49 days [40 µCi of 211At] vs. 31 days [10 µCi of 211At] vs. 21 days [Ctrl]; P Conclusion: Our data support the further development of 211At-CD123 RIT for the treatment of patients with acute leukemia and other CD123+ hematologic malignancies. Figure 1 Figure 1. Disclosures Green: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Cellectar Biosciences: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; JANSSEN Biotech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Therapeutics: Patents & Royalties, Research Funding; Legend Biotech: Consultancy; Neoleukin Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; SpringWorks Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

// George S. Laszlo 1 , Kimberly H. Harrington 1 , Chelsea J. Gudgeon 1 , Mary E. Beddoe 1 , Matthew P. Fitzgibbon 2 , Rhonda E. Ries 1 , Jatinder K. Lamba 3 , Martin W. McIntosh 2 , Soheil Meshinchi 1, 4, 5 , Roland B. Walter 1, 6, 7 1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 2 Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 3 Department of Pharmacotherapy and Translational Research College of Pharmacy, University of Florida, Gainesville, FL, USA 4 Children’s Oncology Group, Arcadia, CA, USA 5 Department of Pediatrics, University of Washington, Seattle, WA, USA 6 Department of Medicine, Division of Hematology, University of Washington, Seattle, WA, USA 7 Department of Epidemiology, University of Washington, Seattle, WA, USA Correspondence to: Roland B. Walter, email: rwalter@fredhutch.org Keywords: acute myeloid leukemia, antigen, CD33, immunotherapy, splice variants Received: April 28, 2016 Accepted: May 17, 2016 Published: May 27, 2016 ABSTRACT With the demonstration of improved survival of some acute myeloid leukemia (AML) patients with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), CD33 has been validated as a target for antigen-specific immunotherapy. Since previous studies identified a CD33 splice variant missing exon 2 (CD33 ∆E2 ) and, consequently, the immune-dominant membrane-distal V-set domain, we investigated the expression and functional characteristics of CD33 transcript variants in AML. In primary AML specimens, we not only found full-length CD33 (CD33 FL ) and CD33 ∆E2 but also corresponding variants containing an alternate exon 7 predicted to encode a CD33 protein lacking most of the intracellular domain (CD33 E7a and, not previously described, CD33 ∆E2,E7a ) in almost all cases. In acute leukemia cell sublines engineered to express individual CD33 splice variants, all splice variants had endocytic properties. CD33 FL and CD33 E7a mediated similar degrees of GO cytotoxicity, whereas CD33 ∆E2 and CD33 ∆E2,E7a could not serve as target for GO. Co-expression of CD33 ∆E2 did not interfere with CD33 FL endocytosis and did not impact CD33 FL -mediated GO cytotoxicity. Together, our findings document a greater-than-previously thought complexity of CD33 expression in human AML. They identify CD33 variants that lack exon 2 and are not recognized by current CD33-directed therapeutics as potential target for future unconjugated or conjugated antibodies.

© 2018 Elsevier Ltd Orientation of cell division plane plays a crucial role in morphogenesis and regeneration. Misoriented cell division underlies many important diseases, such as cancer. Studies with Drosophila and C. elegance models show that Src, a proto-oncogene tyrosine-protein kinase, is a critical regulator of this aspect of mitosis. However, the role for Src in controlling cell division orientation in mammalian cells is not well understood. Using genetic and pharmacological approaches and two extracellular signals to orient cell division, we demonstrated a critical role for Src. Either knockout or pharmacological inhibition of Src would retain the fidelity of cell division orientation with the long-axis orientation of mother cells. Conversely, re-expression of Src would decouple cell division orientation from the pre-division orientation of the long axis of mother cells. Cell division orientation in human breast and gastric cancer tissues showed that the Src activation level correlated with the degree of mitotic spindle misorientation relative to the apical surface. Examination of proteins associated with cortical actin revealed that Src activation regulated the accumulation and local density of adhesion proteins on the sites of cell-matrix attachment. By analyzing division patterns in the cells with or without Src activation and through use of a mathematical model, we further support our findings and provide evidence for a previously unknown role for Src in regulating cell division orientation in relation to the pre-division geometry of mother cells, which may contribute to the misoriented cell division.

Purpose: Exploratory gene expression array analyses suggested multimerin-1 (MMRN1) to be a predictive biomarker in acute myelogenous leukemia (AML). Following up on these studies, we evaluated the role of MMRN1 expression as outcome predictor in two recent Children's Oncology Group trials. Experimental Design: We retrospectively quantified MMRN1 expression in 183 participants of AAML03P1 and 750 participants of AAML0531 by reverse-transcriptase PCR and correlated expression levels with disease characteristics and clinical outcome. Results: In AAML03P1, the highest quartile of MMRN1 expression (expression ≥0.5 relative to β-glucuronidase; n = 45) was associated with inferior event-free survival (EFS; P < 0.002) and higher relapse risk (P < 0.004). In AAML0531, in which we quantified MMRN1 mRNA for validation, patients with relative MMRN1 expression ≥0.5 (n = 160) less likely achieved remission (67% vs. 77%, P = 0.006), and more frequently had minimal residual disease (43% vs. 24%, P = 0.001) after one induction course. They had inferior overall survival (OS; 44% ± 9% vs. 69% ± 4% at 5 years; P < 0.001) and EFS (32% ± 8% vs. 54% ± 4% at 5 years; P < 0.001) and higher relapse risk (57% ± 10% vs. 35% ± 5% at 5 years; P < 0.001). These differences were partly attributable to the fact that patients with high MMRN1 expression less likely had cytogenetic/molecular low-risk disease (P < 0.001) than those with low MMRN1 expression. Nevertheless, after multivariable adjustment, high MMRN1 expression remained statistically significantly associated with shorter OS (HR, 1.57; 95% confidence interval, 1.17–2.12; P = 0.003) and EFS (HR, 1.34; 1.04–1.73; P = 0.025), and higher relapse risk (HR, 1.40; 1.01–1.94; P = 0.044). Conclusions: Together, our studies identify MMRN1 expression as a novel biomarker that may refine AML risk stratification. Clin Cancer Res; 21(14); 3187–95. ©2015 AACR.

CD33 is a myeloid differentiation antigen with endocytic properties. It is broadly expressed on acute myeloid leukemia (AML) blasts and, possibly, some leukemic stem cells and has therefore been exploited as target for therapeutic antibodies for many years. The improved survival seen in many patients when the antibody-drug conjugate, gemtuzumab ozogamicin, is added to conventional chemotherapy validates this approach. However, many attempts with unconjugated or conjugated antibodies have been unsuccessful, highlighting the challenges of targeting CD33 in AML. With the development of improved immunoconjugates and CD33-directed strategies that harness immune effector cells, therapeutics with enhanced efficacy may soon become available. Toxic effects on normal hematopoietic cells may increase in parallel with this increased efficacy and demand new supportive care measures, including possibly rescue with donor cells, to minimize morbidity and mortality from drug-induced cytopenias and to optimize treatment outcomes with these agents in patients with AML.

CD33 is a valid target for acute myeloid leukemia (AML) but has proven challenging for antibody-drug conjugates. Herein, we investigated the cellular determinants for the activity of the novel CD33/CD3-directed bispecific T-cell engager antibody, AMG 330. In the presence of T cells, AMG 330 was highly active against human AML cell lines and primary AML cells in a dose- and effector to target cell ratio-dependent manner. Using cell lines engineered to express wild-type CD33 at increased levels, we found a quantitative relationship between AMG 330 cytotoxicity and CD33 expression; in contrast, AMG 330 cytotoxicity was neither affected by common CD33 single nucleotide polymorphisms nor expression of the adenosine triphosphate-binding cassette (ABC) transporter proteins, P-glycoprotein or breast cancer resistance protein. Unlike bivalent CD33 antibodies, AMG 330 did not reduce surface CD33 expression. The epigenetic modifier drugs, panobinostat and azacitidine, increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity. These findings demonstrate that AMG 330 has potent CD33-dependent cytolytic activity in vitro, which can be further enhanced with other clinically available therapeutics. As it neither modulates CD33 expression nor is affected by ABC transporter activity, AMG 330 is highly promising for clinical exploration as it may overcome some limitations of previous CD33-targeted agents.

BACKGROUND: With gemtuzumab ozogamicin (GO; targeting CD33) and inotuzumab ozogamicin (IO; targeting CD22), 2 antibody-drug conjugates delivering a toxic calicheamicin (CLM) derivative have recently been approved for the treatment of people with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), respectively. While effective in some, many patients do not benefit from these ADCs. It is unclear to what degree anti-apoptotic BCL-2 family members are involved in modulating efficacy of CLM-based ADCs, with limited studies coming to differing conclusions. Given the clinical availability of small molecule inhibitors for BCL-2 family proteins (BCLi), here we clarify the impact of BCL-2 family proteins on the anti-leukemic activity of CLM-ADCs. MATERIALS AND METHODS: Human AML and ALL cell lines were engineered to overexpress BCL-2, BCL-XL, and MCL-1 via lentiviral gene transfer. AML and ALL cell lines as well as AML patient samples were exposed to increasing concentrations of GO or IO with or without the BCL-2 inhibitor ABT-199 (venetoclax), the BCL-2/BCL-XL inhibitor ABT-263 (navitoclax), and the MCL-1 inhibitor AZD5991. Dead cells were enumerated by flow cytometry via 4',6-diamidino-2-phenylindole staining after 72 hours. For BH3 profiling of AML patient specimens, thawed AML patient specimen aliquots were exposed to JC-1 mitochondrial dye and BH3 peptides, and peptide-induced depolarization was then calculated as a percent relative to a CCCP positive control, yielding a priming score for each BH3 peptide. RESULTS: At a dose of 1000 pg/ml, GO killing of ML-1 (AML) cells decreased from 56±5% (mean±SEM) in parental cells to 32±7% (p CONCLUSIONS: Our studies establish an important role of anti-apoptotic BCL-2 family members as resistance factor for CLM-based ADC therapy of acute leukemia. These findings provide the rationale to explore the combination of small-molecule inhibitors of BCL-2 family members with CLM-ADCs as a combination strategy in the clinic to improve the efficacy of GO and, particularly, IO. These therapeutic strategies may incorporate the assessment of the relative contribution of specific BCL-2 family members to an individual cancer patient's disease. Disclosures Jean: Eutropics Pharmaceuticals: Employment. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership. Walter:Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; New Link Genetics: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy.

While survival for patients with acute myeloid leukemia (AML) has increased in recent years, relapse and treatment-related mortality remain a significant problem, especially in older patients. Although the use of gemtuzumab ozogamicin (GO) has validated the concept of antibody directed therapy for AML targeting CD33, complications from myelosuppression and opportunistic infections due to on-target toxicity to normal CD33+ hematopoietic cells have limited this approach. In addition, only a subset of patients derives long-term benefit from GO. Thus, to improve the outcomes of CD33-targeted immunotherapy, more potent drugs are needed such as CD33/CD3 bispecific T-cell Engagers (BiTEs), as are strategies to protect normal blood cells from the cytotoxic effects of these agents. In this study, we assessed the in vivo activity of the clinically exploited CD33/CD3 BiTE AMG330 using our previously described MATCH mouse model (Haworth et al., MTM 2017), in which human CD34+ stem/progenitor cells (HSPCs) are transplanted into neonate mice. This model enables the in vivo maturation of human HSPCs to establish a fully reconstituted blood and immune system, and in particular functional T cells that permit the testing of BiTEs without risks of graft-versus-host disease. Since CD33 is expressed on normal myeloid cells, causing significant on-target/off-leukemia effects, we also investigated whether our previously validated CRISPR/Cas9-based strategy (Humbert et al., Leukemia 2019) protects hematopoiesis from AMG330 activity. Mice were engrafted with mock-(Cas9 only) or CRISPR/Cas9-edited human CD34+ HSPCs to ablate CD33 expression. Normal multilineage engraftment was observed in both experimental groups, resulting in hematopoiesis that either expressed or almost entirely lacked CD33. Mice were also infused with AML tumor cells to validate AMG330 activity, which was administered intravenously to the animals for 5 consecutive days at 0.2mg/kg. AMG330 rapidly eliminated CD33+ Molm14 tumor cells within 5 days (Figure 1A) and reduced the number of circulating CD14+ monocytes in the mock group (Figure 1B), thus confirming the functionality of in vivo-matured autologous T-lymphocytes generated. In contrast, CRISPR/Cas9-edited cells were fully protected from AMG330 cytotoxicity, as evidenced by steady CD14+ counts pre- and post-treatment (Figure 1B). Together, these results validate the MATCH model for testing the efficacy and toxicity of CD33-directed immunotherapies, in particular novel BiTEs. In addition, the combination of HSPC protection and improved CD33 targeting to more effectively kill AML should be of great value for the development of novel BiTEs and other bispecific antibodies for the treatment of AML. Figure 1: A) Leukemia burden measured by luciferase expression in mice from the two experimental groups treated or not with AMG330. B) Quantification of CD14+/CD33+ and CD14+/CD33− cells from peripheral blood of AMG330-treated animals in Cas9 only (n=3) and CRISPR/Cas9 (n = 3) groups. In A) and B), 'pre' is before AMG330 treatment and 'post' is 5 days after administration. Figure 1 Disclosures Walter: New Link Genetics: Consultancy; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Boehringer Ingelheim: Consultancy; Aptevo Therapeutics: Consultancy, Research Funding; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Agios: Consultancy; Amgen: Consultancy. Kiem:Magenta Therapeutics: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; CSL Behring: Consultancy.

Acute myeloid leukemia (AML) encompasses a heterogeneous group of diseases, and novel biomarkers for risk refinement and stratification are needed to optimize patient care. To identify novel risk factors, we performed transcriptome sequencing on 68 diagnostic AML samples and identified 2 transcript variants (-E2 and -E2/3) of the α-subunit (ITGA5) of the very late antigen-5 integrin. We then quantified expression of ITGA5 and these splice variants in specimens from participants of the AAML03P1 trial. We found no association between ITGA5 expression and clinical outcome. In contrast, patients with the highest relative expression (Q4) of the -E2/3 ITGA5 splice variant less likely had low-risk disease than Q1-3 patients (21% vs. 38%, P = 0.027). Q4 patients had worse response to chemotherapy with a higher proportion having persistent minimal residual disease (50% vs. 23%, P = 0.003) and inferior overall survival (at 5 years: 48% vs. 67%, P = 0.015); the latter association was limited to low-risk patients (Q4 vs. Q1-3: 56% vs. 85%, P = 0.043) and was not seen in standard-risk (51% vs. 60%, P = 0.340) or high-risk (33% vs. 38%, P = 0.952) patients. Our exploratory studies indicate that transcriptome sequencing is useful for biomarker discovery, as exemplified by the identification of ITGA5 -E2/3 splice variant as potential novel adverse prognostic marker for low-risk AML that, if confirmed, could serve to further risk-stratify this patient subset.

SummarySrc is a nonreceptor tyrosine kinase that coordinates responses to diverse soluble and adhesive signaling molecules and regulates cell proliferation, survival, differentiation and migration [1]. Normally, Src activity is tightly regulated, and Src-catalyzed phosphorylation is counterbalanced by phosphotyrosine phosphatases [2, 3]. However, deregulated mutant Src causes malignant transformation when highly expressed [4]. Src transformation is dose dependent, but it has been unclear how much mutant Src, compared with endogenous Src, is required for transformation [5]. Here, we show that transformation requires high-level overexpression of mutant src mRNA, in part because active Src protein is degraded by ubiquitin-mediated proteolysis [6]. We show that active but not inactive Src protein is downregulated depending on the putative tumor suppressor and E3 ubiquitin ligase component, Cullin-5 (Cul5). Cul5 removal synergizes with physiological levels of mutant src mRNA to increase protein tyrosine phosphorylation, induce morphological transformation, and deregulate growth. Cul5 also represses Src-induced tumorigenesis and regulates Src signaling in normal cells. These results suggest that, when Src is activated by mutation or physiological mechanisms, its effects are limited by Cul5, which downregulates active Src and its phosphorylated substrates. These findings demonstrate the importance of a new mechanism that downregulates Src signaling in cells.

Background Recent studies have identified myocyte enhancer factor 2C (MEF2C) as cooperating oncogene in acute myeloid leukemia (AML) and suggested a contribution to the aggressive nature of at least some subtypes of AML, raising the possibility that MEF2C could serve as marker of poor-risk AML and, therefore, have prognostic significance. Methods To test this hypothesis, we retrospectively quantified MEF2C expression in pretreatment bone marrow specimens in participants of the AAML0531 trial by reverse-transcriptase polymerase chain reaction and correlated expression levels with disease characteristics and clinical outcome. Results In all 751 available patient specimens, MEF2C messenger RNA (mRNA) was detectable and varied >3000-fold relative to β-glucuronidase. Patients with the highest relative MEF2C expression (4th quartile) less likely achieved a complete remission after one course of chemotherapy than the other patients (67 vs. 78 %, P = 0.005). They also had an inferior overall survival (P = 0.014; at 5 years 55 ± 8 vs. 67 ± 4 %), inferior event-free survival (P

Treatment of neuronal cells with leukemia inhibitory factor (LIF) results in increased M(2) muscarinic acetylcholine receptor promoter activity. We demonstrate here that multiple promoter elements mediate LIF stimulation of M(2) gene transcription. We identify a LIF inducible element (LIE) in the M(2) promoter with high homology to a cytokine-inducible ACTG-containing sequence in the vasoactive intestinal peptide promoter. Mutagenesis of both a STAT (signal transducers and activators of transcription) element and the LIE in the M(2) promoter is required to attenuate stimulation of M(2) promoter activity by LIF completely. Mobility shift assays indicate that a LIF-stimulated complex binds to a 70 base pair M(2) promoter fragment. Furthermore, a STAT element within this fragment can bind to LIF-stimulated nuclear STAT1 homodimers in vitro. Mutagenesis experiments show that cytokine-stimulated activation of M(2) promoter activity requires tyrosine residues on glycoprotein 130 (gp130) that are also required for both STAT1 and STAT3 activation. Dominant negative STAT1 or STAT3 can block LIF-stimulated M(2) promoter activity. Real-time RT-PCR analysis indicates that LIF-stimulated induction of M(2) mRNA is partially dependent on protein synthesis. These results show that regulation of M(2) gene transcription in neuronal cells by LIF occurs through a complex novel mechanism that is dependent on LIE, STAT and de novo protein synthesis.

Members of the interleukin-6 (IL-6) family of cytokines exert their biological effects via binding to their cognate ligand-binding receptor subunit on a target cell. The subsequent recruitment of the common signal transducer glycoprotein 130 and activation of the JAK/STAT and SHP-2/Ras/mitogen-activated protein kinase (MAPK) pathways are responsible for the majority of cellular responses elicited by IL-6 cytokines. Several types of experiments suggest that the Src family of kinases (SFK) also participates in IL-6 family cytokine-mediated signaling events. SYF cells, which lack expression of SFKs Src, Yes, and Fyn, were used to determine the role of SFKs in IL-6 family cytokine signaling and gene induction. SYF and wild type (WT) control fibroblasts displayed similar activation of signaling intermediates following stimulation with leukemia inhibitory factor (LIF). LIF-stimulated tyrosine phosphorylation of SHP-2 and subsequent activation of MAPK in SYF cells were identical to that seen in LIF-stimulated WT cells. Both LIF-stimulated tyrosine phosphorylation of STAT1 and STAT3, as well as LIF-stimulated DNA binding activity of STAT-containing nuclear complexes were indistinguishable when compared in SYF and WT cells. In addition, the phosphatidylinositol 3-kinase-sensitive Akt kinase and p38 MAPK were activated by LIF in both SYF and WT cells. Furthermore, LIF-stimulated expression of c-fos, egr-1, and suppressor of cytokine signaling-3 was retained in SYF cells. The IL-6 family cytokine oncostatin M was also capable of activating MAPK, STAT3, STAT1, Akt, and p38 in both WT and SYF cells. These results demonstrate that IL-6 family cytokines can activate a full repertoire of signaling pathways and induce gene expression independent of SFKs.

Neutrophil elastase, a serine protease of the chymotrypsin family encoded by ELANE (ELA2), is primarily found in azurophilic (primary) granules of neutrophils [1,2]. Physiologically, neutrophil ela...

Deregulated cytokine signaling is a characteristic feature of acute myeloid leukemia (AML), and expression signatures of cytokines and chemokines have been identified as a significant prognostic factor in this disease. Given this aberrant signaling, we hypothesized that expression of suppressor of cytokine signaling-2 (SOCS2), a negative regulator of cytokine signaling, might be altered in AML and could provide predictive information. Among 188 participants of the Children's Oncology Group AAML03P1 trial, SOCS2 mRNA levels varied6000-fold. Higher (median) SOCS2 expression was associated with inferior overall (60 ± 10% vs. 75 ± 9%, p = 0.026) and event-free (44 ± 10% vs. 59 ± 10%, p = 0.031) survival. However, these differences were accounted for by higher prevalence of high-risk and lower prevalence of low-risk disease among patients with higher SOCS2 expression, limiting the clinical utility of SOCS2 as a predictive marker. It remains untested whether high SOCS2 expression identifies a subset of leukemias with deregulated cytokine signaling that could be amenable to therapeutic intervention.

Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML), but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34(+)/CD33(-) cells revealed polyclonal growth in highly curable AMLs, suggesting that mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34(+)/CD33(-) cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications.

Antibodies have created high expectations for effective yet tolerated therapeutics in acute myeloid leukemia (AML). Hitherto the most exploited target is CD33, a myeloid differentiation antigen found on AML blasts in most patients and, perhaps, leukemic stem cells in some. Treatment efforts have focused on conjugated antibodies, particularly gemtuzumab ozogamicin (GO), an anti-CD33 antibody carrying a toxic calicheamicin-g 1 derivative that, after intracellular hydrolytic release, induces DNA strand breaks, apoptosis, and cell death. Serving as paradigm for this strategy, GO was the first anti-cancer immunoconjugate to obtain regulatory approval in the U.S. While efficacious as monotherapy in acute promyelocytic leukemia (APL), GO alone induces remissions in less than 25-35% of non-APL AML patients. However, emerging data from well controlled trials now indicate that GO improves survival for many non-APL AML patients, supporting the conclusion that CD33 is a clinically relevant target for some disease subsets. It is thus unfortunate that GO has become unavailable in many parts of the world, and the drug's usefulness should be reconsidered and selected patients granted access to this immunoconjugate.

Gemtuzumab ozogamicin (GO), an immunoconjugate between an anti-CD33 antibody and a calicheamicin-γ(1) derivative, induces remissions and improves survival in a subset of patients with acute myeloid leukemia (AML). As the mechanisms underlying GO and calicheamicin-γ(1) resistance are incompletely understood, we herein used flow cytometry-based single cell network profiling (SCNP) assays to study cellular responses of primary human AML cells to GO. Our data indicate that the extent of DNA damage is quantitatively impacted by CD33 expression and drug efflux activity. However, although DNA damage is required for GO-induced cytotoxicity, it is not sufficient for effective cell kill, suggesting that downstream anti-apoptotic pathways may function as relevant resistance mechanisms. Supporting this notion, we found activated PI3K/AKT signaling to be associated with GO resistance in vitro in primary AML cells. Consistently, the investigational AKT inhibitor MK-2206 significantly sensitized various human AML cells to GO or free calicheamicin-γ(1) with particularly pronounced effects in otherwise GO or free calicheamicin-γ(1)-resistant cells. Likewise, MK-2206 also sensitized primary AML cells to calicheamicin-γ(1). Together, our findings illustrate the capacity of SCNP assays to discover chemotherapy-related biological pathways and signaling networks relevant to GO-induced genotoxic stress. The identification of AKT signaling as being associated with GO resistance in vitro may provide a novel approach to improve the in vivo efficacy of GO/calicheamicin-γ(1) and, by extrapolation, other DNA damage-based therapeutics.

Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5-RING-E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src-Cas-induced ruffling through SOCS6.

The cytokines that signal through the leukemia inhibitory factor (LIF) receptor are members of the neuropoietic cytokine family and have varied and numerous roles in the nervous system. In this report we have determined the effects of growth factor stimulation on LIF receptor (LIFR) expression and signal transduction in the human neuroblastoma cell line NBFL. We show here that stimulation of NBFL cells with either epidermal growth factor or fibroblast growth factor decreases the level of LIFR in an extracellular signal-regulated kinase (Erk)1/2-dependent manner and that this downregulation is due to an increase in the apparent rate of lysosomal LIFR degradation. Growth factor-induced decreases in LIFR level inhibit both LIF-stimulated phosphorylation of signal transducers and activators of transcription 3 (STAT3) and LIFR-mediated gene induction. We also show that Ser1044 of LIFR, which we have previously shown to be phosphorylated by Erk1/2, is required for the inhibitory effects of growth factors. Neurons are exposed to varying combinations and concentrations of growth factors and cytokines that influence their growth, development, differentiation and repair in vivo. These findings demonstrate that LIFR expression and signaling in neuroblastoma cells can be regulated by growth factors that are potent activators of the mitogen activated protein kinase pathway, and thus illustrate a fundamental mechanism that underlies cross-talk between receptor tyrosine kinase and neuropoietic cytokine signaling pathways.

Background: Myocyte enhancer factor 2C (MEF2C) was initially identified as essential transcription factor for cardiac muscle development. However, subsequent studies have indicated that MEF2C plays a much broader biological role, including in the normal hematopoietic system. Recent studies have now identified MEF2C as cooperating oncogene in acute myeloid leukemia (AML) and suggested a contribution to the aggressive nature of at least some subtypes of AML. These findings raised the possibility that MEF2C could serve as marker of poor-risk disease and, therefore, have prognostic significance in AML. To test this hypothesis, we retrospectively quantified MEF2C expression in participants of the AAML0531 trial and correlated expression levels with disease characteristics and clinical outcome. Patients and Methods: AAML0531 (NCT00372593) was a multicenter phase 3 study that determined the addition of gemtuzumab ozogamicin to intensive chemotherapy among 1,022 eligible patients aged Results: In all 751 available patient specimens, MEF2C mRNA was detectable and varied >3,000-fold relative to GUSB (0.0091-29.1272 [median: 0.7978]). Patients with the highest relative MEF2C expression (4th quartile) less likely achieved a complete remission after one course of chemotherapy than the other patients (67% vs. 78%, P=0.005). They also had an inferior overall survival (P=0.014; at 5 years: 55±8% vs. 67±4%), inferior event-free survival (P Conclusion: High MEF2C expression identifies a subset of AML patients with adverse-risk disease features and poor outcome. These findings provide the rationale for therapeutic targeting of MEF2C transcriptional activation in AML. Disclosures Walter: AstraZeneca, Inc.: Consultancy; Covagen AG: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen, Inc.: Research Funding; Pfizer, Inc.: Consultancy; Amphivena Therapeutics, Inc.: Consultancy, Research Funding.

The CD33/CD3-bispecific T-cell engaging (BiTE) antibody construct, AMG 330, potently lyses CD33+ leukemic cells in vitro. Using specimens from 41 patients with acute myeloid leukemia (AML), we studied the factors that might contribute to clinical response or resistance. For this purpose, thawed aliquots of primary AML samples were immunophenotypically characterized and subjected to various doses of AMG 330 in the presence or absence of healthy donor T-cells. After 48 hours, drug-specific cytotoxicity was quantified and correlated with CD33 expression levels, amounts of T-cells present, and other disease characteristics. AMG 330 caused modest cytotoxicity that was correlated with the amount of autologous T-cells (P = 0.0001) but not CD33 expression, as AMG 330 exerted marked cytotoxic effects in several specimens with minimal CD33 expression. With healthy donor T-cells added, AMG 330 cytotoxicity depended on the drug dose and effector:target (E:T) cell ratio. High cytotoxic activity was observed even with minimal CD33 expression, and AMG 330 cytotoxicity and CD33 expression correlated only at high E:T cell ratio and high AMG 330 doses (P

Preclinical and emerging clinical studies demonstrate that bispecific T-cell engaging (BiTE) antibody constructs can potently lyse targeted tumor cells, but the determinants for their activity remain incompletely understood. Using human acute myeloid leukemia (AML) cell lines engineered to overexpress individual T-cell ligands, we found that expression of the inhibitory ligands, PD-L1 and PD-L2, reduced the cytolytic activity of the BiTE antibody construct targeting CD33, AMG 330; conversely, expression of the activating ligands, CD80 and CD86, augmented the cytotoxic activity of AMG 330. Consistent with these findings, treatment with an activating antibody directed at the co-stimulatory T-cell receptor, CD28, significantly increased AMG 330-induced cytotoxicity in human AML cell lines. Using specimens from 12 patients with newly diagnosed or relapsed/refractory AML, we found that activation of CD28 also increased the activity of AMG 330 in primary human AML cells (P=0.023). Together, our findings indicate that T-cell ligands and co-receptors modulate the anti-tumor activity of the CD33/CD3 BiTE antibody construct, AMG 330. These findings suggest that such ligands/co-receptors could serve as biomarkers of response and that co-treatment strategies with pharmacological modulators of T-cell receptor signaling could be utilized to further enhance the activity of this targeted therapeutic.

A targeted disruption of the RIalpha isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RIalpha is the only isoform that is essential for early embryonic development. RIalpha homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RIalpha mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RIalpha mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RIalpha mutants to mice carrying a targeted disruption in the Calpha catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RIalpha is responsible for the phenotype. Primary embryonic fibroblasts from RIalpha mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.

Background: Both cytogenetic and molecular abnormalities have been identified in acute myeloid leukemia (AML) that provide the framework for diagnostic classification and risk-stratification schemes. However, there are currently only a small number of informative prognostic markers, and it is a recurrent clinical observation that this limited battery fails to accurately predict outcome for many patients. This highlights the need for additional and refined tools to characterize disease risk in AML. Patients and Methods: For gene discovery, gene expression array data from 211 pediatric patients with AML were queried. Subsequently, expression of the gene of interest was quantified in cryopreserved pretreatment specimens from patients enrolled on the AAML03P1 and AAML0531 trials: AAML03P1 was a pilot phase 3 study investigating the feasibility of combining gemtuzumab ozogamicin (GO) with intensive chemotherapy in pediatric patients up to age 30, whereas AAML0531 was a phase 3 study on >1,000 patients testing the value of GO addition to chemotherapy in a randomized fashion. Taqman-based quantitative reverse-transcriptase PCR was used to quantify mRNA expression of the gene of interested and the housekeeping gene, b-glucuronidase (GUSB). Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa), high risk (-5/5q-, monosomy 7, or FLT3-ITD with high allelic ratio), or standard risk (all other patients with cytogenetic/molecular data). Results: Multimerin-1 (MMRN1), a member of the elastin microfibrillar interface protein (EMILIN)/multimerin family that may mediate cellular adhesion via integrin receptors, was identified in gene expression array data as a gene whose expression varied widely, with expression levels possibly related to patient outcomes. To study this possible relationship further, we quantified MMRN1 mRNA in 183 participants of AAML03P1 and correlated expression levels with clinical outcome. MMRN1 expression varied >80,000-fold relative to GUSB. Patients with the highest MMRN1 expression (4th quartile, corresponding to a relative MMRN1 expression of >0.5) indeed had inferior event-free survival (EFS; P130,000-fold relative to GUSB. Using a cut-off for high MMRN1 expression as defined in the AAML03P1 cohort (i.e. relative MMRN1 Conclusion: This study identifies high MMRN1 expression as a novel adverse prognostic factor in pediatric AML. Although the association between adverse outcome and high MMRN1 expression is at least partly due to the higher proportion of adverse-risk features among AML patients with high MMRN1 expression, high MMRN1 expression continued to be associated with inferior survival even after adjustment of disease risk. Disclosures No relevant conflicts of interest to declare.

Background: Recent studies have demonstrated that AMG 330, a novel CD33/CD3-directed bispecific T-cell engaging (BiTE¨) antibody, is highly active against CD33+ AML cell lines and can potently lyse leukemic blasts from AML patients. Here, we have investigated the preclinical determinants for AMG 330 activity in primary newly diagnosed and relapsed/refractory human AML samples to prospectively understand factors that might contribute to clinical response or resistance. Patients and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML who consented to their use for research purposes. CD33 expression on myeloblasts (identified by appropriate CD45 and side-scatter properties) and the percentage of endogenous CD3+ T-cells were quantified by flow cytometry. To determine drug-induced cytotoxicity, AML cells were incubated in culture medium containing various concentrations of AMG 330; in some experiments, exogenous T-cells (isolated via magnetic cell sorting from a healthy adult volunteer who underwent leukapheresis) were labeled with CellVue Burgundy and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; AML cells were identified by forward/side scatter properties and negativity for CellVue Burgundy dye. Specific drug-induced cytotoxicity was calculated as: 100 x (1 – live target cellstreated/live target cellscontrol), and presented as mean±SEM. Results: Forty-one of the 49 studied specimens were included in our analyses as they had >40% myeloblasts upon thaw and were >50% and >30% viable at baseline and after 48 hours, respectively. Median age of the patients was 65.3 (range: 23.9-80.0) years. The median percentage of myeloid blasts and CD3+ T-cells in the studied specimens was 87.1% (range: 55.1-97.0%) and 2.0% (range: 0-27.3%), respectively, and median sample viability after 48 hours in culture was 76.7% (range: 31.1-93.5%). These characteristics were similar between the newly diagnosed (n=21) and relapsed/refractory (n=20) AML specimens. In the absence of healthy donor T-cells, AMG 330 resulted in modest cytotoxicity (at 500 pg/mL: 11.8±2.5%) that was correlated with the amount of autologous T-cells (at 500 pg/mL; r=0.566; p=0.0001; Spearman's rank correlation test). On the other hand, AMG 330 exerted marked cytotoxic activity in several specimens with very low CD33 expression, and there was no correlation between AMG 330 cytotoxicity and CD33 expression (at 500 pg/mL; r=-0.048; p=0.77). In the presence of healthy donor T-cells, AMG 330 cytotoxicity was strictly dependent on the drug dose (e.g. p Conclusion: AMG 330 causes potent cytolysis of primary human AML cells in vitro in a dose- and E:T cell ratio-dependent manner across the entire spectrum of cytogenetic/molecular risk and disease stage, even in specimens with very low expression of CD33. Lower activity of AMG 330 was observed in relapsed/refractory AML specimens (relative to newly diagnosed AML specimens) and intermediate- or adverse-risk disease specimens (relative to favorable-risk specimens) suggesting the potential presence of yet undefined, CD33-independent, relative resistance mechanisms in defined patient subsets. Figure 1 Figure 1 Figure 1. Figure 2 Figure 2 Figure 2. Disclosures Off Label Use: Use of AMG 330 in AML (AMG 330 is investigational drug). Newhall:Amgen, Inc: Employment. Sinclair:Amgen, Inc: Employment. Frankel:Amgen, Inc: Employment. Kischel:Amgen, Inc: Employment. Chen:Amgen, Inc: Employment. Walter:Amgen, Inc: Research Funding; Seattle Genetics, Inc.: Research Funding; Amphivena Therapeutics, Inc.: Consultancy; Covagen AG: Consultancy.

Background The cellular origin of human acute myeloid leukemia (AML) remains controversial. Limited G6PD-based X chromosome inactivation (XCI) studies suggested heterogeneity of leukemic stem cells (LSCs), with evidence of some AMLs resulting from multipotent CD34+/CD33- stem cells and others emerging from, or predominantly involving, lineage-committed CD34+/CD33+ myeloid precursors. So far, the difficulty of growing stem/progenitor cells from primary AML specimens has provided a major challenge for precise study of human LSCs and clinical implications of their heterogeneity. To overcome this, we developed a novel co-culture method to support long-term in vitro growth of primary AML specimens together with a highly sensitive XCI assay for isolated immature progenitor cells. Methods Diagnostic bone marrow specimens from 50 adult females with newly diagnosed AML were obtained from the SWOG Sample Repository. All patients received standard induction therapies without the CD33-targeting immunoconjugate, gemtuzumab ozogamicin. Human umbilical vein endothelial cells, transduced with a lentiviral construct encoding the open reading frame 1 of the early region 4 of adenovirus to provide growth factor/serum-independence, were co-cultured with aliquots of flow cytometrically-isolated CD34+/CD33- and CD34+/CD33+ cells in medium containing the aryl hydrocarbon receptor antagonist, StemReginin-1. Cultures were replenished with fresh medium weekly and kept in hypoxia for up to 8 weeks, and defined fractions plated after 4, 6, and 8 weeks for colony-forming cell assays. Resulting colony forming units-granulocyte and/or macrophage (CFU-GMs) were harvested and individually assessed for XCI via methylation-sensitive PCR-based analysis of a polymorphic short tandem repeat (STR) in the X-linked human androgen-receptor gene (HUMARA). Specimen growth was considered if it yielded ≥5 CFU-GMs after ≥4 weeks of co-culture. Specimens with >75% disease-specific CFU-GMs were considered clonal. Results Among the 50 specimens, 45 (90%) were informative with regard to HUMARA STR, allowing clonal assessment of isolated cell progeny. Sixteen (35.6%) yielded CFU-GMs derived from CD34+/CD33- cells after long-term (≥4 weeks) co-culture sufficient for clonal analyses, whereas the remaining samples yielded either no growth (n=22) or insufficient CFU-GM growth (n=8) from this starting cell population. Five of the 16 specimens with sufficient CFU-GM growth derived from CD34+/CD33- cells (31.3%) yielded colonies consistent with a polyclonal growth pattern, and 3 samples without sufficient CFU-GM growth derived from CD34+/CD33- cells yielded colonies from unsorted cells that were consistent with a polyclonal growth pattern; these 8 leukemias were cytogenetically classified as favorable (n=3) or intermediate-risk (n=5, including 2 with normal karyotype and NPM1 but not FLT3/ITD mutation). In contrast, 11 specimens yielded CFU-GMs growth derived from CD34+/CD33- with a pattern consistent with clonal growth. The complete remission rate was slightly but statistically non-significantly higher among patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- cells relative to those with clonal progenitor growth (87.5% vs. 63.6%, P=0.34). More importantly, however, the overall and relapse-free survival of patients whose specimens yielded polyclonal CFU-GMs derived from CD34+/CD33- or unsorted cells was significantly longer than that for patients whose specimens yielded a clonal progenitor growth pattern after long-term culture of CD34+/CD33- cells (P=0.0006 and P=0.0054, respectively; Figure 1); restriction of this analysis to the 16 specimens with sufficient CFU-GM growth from CD34+/CD33- cells did not change this outcome difference (e.g. hazard ratio for overall survival for restricted dataset: 0.13 [95% confidence interval: 0.06-0.72] vs 0.08 [0.04-0.44]). Conclusion Our novel co-culture method allows for the study of progenitor/stem cell involvement in a significant portion of human AMLs. Our data suggest that a subset of adult AMLs emerges from, or predominantly involves, only lineage-committed CD34+/CD33+ myeloid precursors but not the less mature CD34+/CD33- precursors. This subset, which is comprised of AMLs with diverse cytogenetic/molecular abnormalities, is characterized by an excellent outcome with intensive chemotherapy. Disclosures: No relevant conflicts of interest to declare.