Primary AbstractAcute Myeloid Leukemia Research Articles

Abstract 2064: Unveiling the correlation between gemtuzumab-ozogamicin efficacy and CD33+ expression in AML primary samples using the novel AML vitroscreen

Abstract Acute myeloid leukemia (AML) poses a significant therapeutic challenge, with a rising incidence in recent times. This clonal hematopoietic disorder of progenitor and stem cells exhibits aggressive behavior, and despite initial remission, recurrence is common1. The conventional shift from chemotherapy to monoclonal antibodies and antibody-drug conjugates (ADCs) has been hindered by the absence of AML-specific antigens. Notably, CD33, expressed strongly in 80-90% of AML cells, has become a target for gemtuzumab-ozogamicin (Mylotarg), the sole FDA approved ADC for AML treatment2. While advancements in understanding AML molecular mechanisms have influenced clinical approaches, the lack of clinically representative models hinders therapeutic progress. Existing models, both in vivo and ex vivo, struggle to capture the heterogeneity and complexity of AML3. Addressing this, we present our proprietary assay, Champions AML VitroScreen®4, designed for testing therapeutic candidates in primary AML samples. Our diverse bank of deeply characterized AML samples encompasses multiple subtypes, allowing evaluation of therapeutic responses through cell viability, proliferation, and clonal composition analyses. To validate our novel assay, we employed the VitroScreen platform to test Mylotarg across various AML subtypes, considering CD33 expression levels. By assessing more than 20 AML models, we determined the IC50 for Mylotarg and observed dose-dependent responses, where sensitivity to the drugh correlated with CD33 expression. Through multiple endpoints, including viability, proliferation, apoptosis, and phenotyping, we categorized primary AML models as responders, low responders, or resistant, establishing a strong positive correlation between Mylotarg sensitivity and CD33 expression. Our innovative approach, utilizing deeply characterized AML patient samples in the AML VitroScreen® platform, facilitates high-throughput drug testing. This system, serving as an ideal clinical translational setting in preclinical research, holds promise for developing tailored therapeutic strategies. Ultimately, this approach could significantly impact patient outcomes by offering improved strategies for both primary and recurrent AML cases. Citation Format: Mara Gilardi, Garima Kaushik, Brandon Walling, Paolo Schiavini, Stefano Cairo, Marianna Zipeto. Unveiling the correlation between gemtuzumab-ozogamicin efficacy and CD33+ expression in AML primary samples using the novel AML vitroscreen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2064.

Abstract 7468: Development of a high complexity flow cytometry panel to assess minimum residual disease in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a highly heterogeneous disease with a diverse range of abnormal myeloid subpopulations resulting in challenges in clinical diagnosis and treatment. Many methods such as flow cytometry are used to provide precise information for risk stratification and treatment methods. Flow cytometry utilizes the expression patterns of cellular markers to characterize the high diversity in AML subpopulations with high sensitivity and specificity. This coupled with rapid analysis timelines allows for flow cytometry to be an excellent tool for the identification of AML lineage and progression through both phenotypic analysis and difference from normal. As AML lineage and disease progression are predictors of residual disease and relapse; the need for effective and accurate characterization of AML populations is critical. Here, Champions Oncology has developed a high-complexity 18-color flow cytometry panel to identify AML subpopulations at high resolution. We have characterized AML samples to evaluate the expression of phenotypic markers and their heterogeneity. Utilizing primary AML samples, we have evaluated the stability of AML subpopulations to understand how delayed analysis, such as what is seen in clinical trials, may impact marker expression and observed key populations were stable for up to 72 hours after collection. In addition to stability, understanding the lower limits of detection is critical for AML patients, particularly for minimum residual disease and relapse screening. The use of a flow cytometric panel again provides an excellent tool for these tests due to the single cell nature that allows for the interrogation of individual populations with primary AML subpopulations detected at a range of 10−3 to 10−4. Finally, with hundreds of never-passaged AML models characterized and cryopreserved by both flow cytometry and sequencing at Champions Oncology, we investigated how the cryopreservation processes impacted the expression of AML subpopulations. We have observed similar expression and detection of AML populations following cryopreservation indicating that these models can be utilized as an excellent tool for further studies. Collectively, our data indicates that the panel developed at Champions Oncology provides robust data analysis of AML subpopulations and can be an excellent tool for the tracking of AML disease progression in the clinic. Citation Format: Jessica Pearl, Elena Bruni, Haoting Hsu, Brandon Walling, Karin Abarca-Heidemann. Development of a high complexity flow cytometry panel to assess minimum residual disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7468.

Abstract 4740: Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy in urgent need of improved therapeutic strategies. AML blasts frequently rely on the anti-apoptotic protein, Bcl-2, for survival. The specific Bcl-2 inhibitor, venetoclax (VEN), is FDA-approved in combination with low-dose cytarabine (LDAC) or azacitidine for patients unfit for intensive induction chemotherapy. Unfortunately, responses are transient due to upregulation of compensatory survival proteins (e.g., Bcl-xL or Mcl-1) or resistance characterized by reprogrammed mitochondrial bioenergetics. Our research aims to improve current AML treatments and understanding of AML pathogenesis by targeting dysregulated sphingolipid metabolism. Ceramides are tumor-suppressor sphingolipids that mediate therapy-induced cell death. Sphingolipid dysregulation that results in decreased ceramide content and/or enhanced ceramide catabolism is an emerging AML hallmark. We’ve previously shown that acid ceramidase (AC), a ceramide catabolizing lysosomal enzyme, is overexpressed in AML and contributes to drug resistance. Here, we demonstrated that AC inhibition enhanced VEN cytotoxicity. Combining the AC inhibitor/ceramide analog, SACLAC, with VEN resulted in synergistic lethality in multiple AML cell lines in cell viability and apoptosis assays. Genetic inhibition of AC also improved VEN cytotoxicity. The SACLAC+VEN combination achieved Bliss scores (SynergyFinder 2.0) between 22 and 32 in cell lines, which indicate a highly synergistic combination. SACLAC+VEN efficacy was comparable to the FDA-approved combination of VEN+LDAC when tested in 67 primary AML patient samples. Pharmacological inhibition of AC also increased the efficacy of the VEN+LDAC combination. Mechanistically, the observed synergistic lethality was independent of changes to Bcl-2, Mcl-1, and Bcl-xL protein levels. Combined AC and Bcl-2 inhibition resulted in synergistic ceramide accumulation, integrated stress response (ISR) activation, and caspase-dependent apoptosis characterized by impaired mitochondrial function. Pretreatment with the pan-caspase inhibitor, zVAD-FMK, or ISR inhibitor, ISRIB, significantly rescued cell death. Ongoing studies aim to further characterize mitochondrial impairment and ISR activation induced by inhibiting AC and Bcl-2. Taken together, these results detail the first report of ISR activation and mitochondrial defects elicited by combined AC and Bcl-2 inhibition as contributory to cytotoxic impact (mechanism) and warrant additional studies of AC inhibitors with frontline AML therapeutics. Citation Format: Johnson Ung, Su-Fern Tan, Jeremy J. Shaw, Todd E. Fox, Maansi Taori, David F. Claxton, Kelsey H. Fisher-Wellman, Myles C. Cabot, David J. Feith, Thomas P. Loughran. Acid ceramidase inhibition enhances venetoclax sensitivity in preclinical models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4740.

Abstract 1703: SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility

Abstract Acute Myeloid Leukemia (AML) is characterized by the unfettered proliferation and poor differentiation of myeloid cells causing reduced survival. The mammalian BAF complexes (also known as SWI/SNF) are important regulators of MYC and the cell cycle. There are three BAF complexes (cBAF, ncBAF and pBAF) that share several subunits. SMARCA2 and 4 are mutually exclusive ATPase subunits within the BAF complexes that use ATP hydrolysis and helicase activity to remodel chromatin via nucleosome sliding and removal. PBRM1 is a SMARCA4 associated factor present in pBAF. The association of SMARCA4 with regulatory elements of transcription factors makes it an intriguing target for AML. To evaluate the role of SMARC2/4 in AML, we analyzed RNA transcripts from highly purified primary AML initiating populations and control samples. Sorted leukemia stem cells (Lin-ve, CD34+, CD38-) overexpressed SMARCA2/4 when compared to healthy cells. A novel and highly selective SMARCA2/4 and PBRM1 PROTAC degrading agent AU-15330 was used to investigate the therapeutic effectiveness of targeting BAF complexes as well as the role of SMARCA2/4 in leukemic pathogenesis. Our data demonstrate that AU-15330 effectively degrades SMARCA2/4 and PBRM1 in human leukemia cell lines with varying mutational profiles; MV4-11 was the most sensitive cell line. When evaluating healthy CD34+ HSPCs as well as patient derived AML cells in colony assays, AU-15330 treatment increased differentiation in patient AML samples with minimal effects on healthy CD34+ cells. A xenograft of patient derived AML cells in irradiated mice was performed to determine the requirement of SMARCA2/4 in leukemia stem cell maintenance. Mice treated with AU-15330 had greater myeloid differentiation, fewer human cells in their bone marrows and reduced leukemia stem cell activity, seen in secondary transplants. To understand the underlying mechanism of how SMARCA2/4 and PBRM1 promote leukemic pathogenesis, we used a multi-omic analysis of transcription and chromatin dynamics in MV4-11 cells: ATAC-seq and RNA-seq identified downregulated genes with decreased accessibility, PRO-seq determined early transcriptional changes and HiChIP showed changes in enhancer promoter loops with SMARC2/4 degradation. Several leukemia related transcription factors as well as inflammatory genes were identified from these analyses as possible targets of the BAF complexes in the context of AML. Finally, to determine the effect of SMARCA2/4 degradation on pBAF association with chromatin, single cell imaging was performed of U2OS cells with HALO-tagged PBRM1 treated with the degrader or DMSO. pBAF in the degrader treated cells improved engagement with target chromatin however, a decrease in the stability of the complex binding to the nucleosome when compared to DMSO treated cells was shown. Taken together, these studies will determine the role of SMARCA2/4 in leukemia disease. Citation Format: Sarah Aminov, Kith Pradhan, Srabani Sahu, Shanisha Gordon-Mitchell, Emma Rabinovich, Ariel Fromowitz, Joyeeta Chakraborty, Srinivas Aluri, Rongbao Zhao, Nayem Haque, Milagros Carbajal, Aditi Shastri, Ulrich Steidl, Susanta Samajdarr, Akhil Kumar, Murali Ramachandra, Robert A. Coleman, Kristy Stengel, Amit K. Verma. SMARCA2/4 degraders relieve the differentiation block in AML via changes in chromatin looping and accessibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1703.

Abstract NG07: Targeting polyamines metabolism in acute myeloid leukemia stem cells

Abstract NG07: Targeting polyamines metabolism in acute myeloid leukemia stem cells

Abstract 2677: ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML

Abstract Acute Myeloid Leukemia (AML) is a rare but catastrophic hematological cancer with a poor prognosis, high mortality, and relapse rates. Several new agents targeting specific genes or cell survival pathways have been recently approved by the FDA. AML treatment with single agents often results in resistance; therefore, combination therapies may provide more durable and effective responses. Rho kinases (ROCK1/2) are aberrantly expressed in AML patient cells, and ROCK1 has emerged lately as a potential therapeutic target for AML treatment. Previously, our group developed a new ROCK inhibitor DJ4 as an effective inducer of AML cell death. Our initial screening of DJ4 with AML standard therapy (cytarabine, daunorubicin, azacytidine, venetoclax) showed promising synergy between DJ4 and the BCL2 (B-cell lymphoma-2) inhibitor venetoclax (VEN). Enrichment of ROCK pathway in recent CRISPR drop-out screening after VEN treatment in MOLM-13 cells further validates the synergistic cooperation between ROCK and BCL2 pathways. Though VEN is widely used in hematological malignancies, its clinical efficacy is limited due to the development of resistance. Thus, we sought to investigate the novel strategy of combining VEN with ROCK inhibitors (ROCKi) for AML treatment. The combination of VEN with ROCKi (Fasudil, Y-27632, GSK269962A, DJ4) exhibited synergetic cytotoxicity in naïve (MOLM-13, MV4-11) and VEN-resistant (MV4-11/VENR, HL-60, OCI-AML3, U937) human AML cell lines. Flow cytometric analysis of Annexin V and cellular ROS levels in MV4-11 (naïve and VENR) cells upon co-treatment with sub-lethal doses of VEN and ROCKi demonstrated a significant (p<0.05) increase in the apoptosis and oxidative stress compared to single agents. Treatment of MV4-11 cells with VEN+ROCKi enhanced the activity of several caspases in the MultiCaspase assay. Furthermore, the colony-forming ability of AML cell lines and primary AML patient cells was significantly reduced with the combination treatment, while single agents exhibited minimal effects. MV4-11 (naïve and VENR) cells displayed alterations in the levels of critical apoptosis regulators (Bcl2, Bax, Mcl1, cleaved caspase-3) upon co-treatment in immunoblotting analysis. Additionally, MV4-11-Luc xenograft mice exhibited a significant reduction in the average leukemic burden after combination therapy relative to single agents (p<0.05). Altogether, our in vitro and in vivo findings established a synergetic interaction between VEN and ROCKi to effectively inhibit AML cell progression and overcome the VEN resistance (inherent or acquired). These results indicate that therapy co-targeting BCL2 and ROCK pathways exerted antileukemic activity by inducing apoptosis and may be particularly effective for AML treatment. Further studies are ongoing to explore the precise molecular mechanism of action of the VEN+ROCKi combination and its efficacy in pre-clinical animal models. Citation Format: Upendarrao Golla, Satyam Patel, Diwakar B. Tukaramrao, Jeremy Hengst, Juliana Restrepo, Shantu Amin, Dhimant Desai, Sinisa Dovat, David Claxton, Arati Sharma. ROCK inhibitors synergize with the BCL2 inhibitor venetoclax for treatment of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2677.

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

Effective Killing of Acute Myeloid Leukemia by TIM-3 Targeted Chimeric Antigen Receptor T Cells.

Acute myeloid leukemia (AML) is an aggressive disease with poor outcomes, overwhelmingly due to relapse. Minimal residual disease (MRD), defined as the persistence of leukemic cells after chemotherapy treatment, is thought to be the major cause of relapse. The origins of relapse in AML have been traced to rare therapy-resistant leukemic stem cells (LSCs) that are already present at diagnosis. Effective treatment strategies for long-term remission are lacking, as it has been difficult to eliminate LSCs with conventional therapy. Here, we proposed a new approach based on the chimeric antigen receptor (CAR)-directed T lymphocytes, targeting T-cell immunoglobulin, and mucin domain 3 (TIM-3) to treat MRD in patients with AML. TIM-3 is selected as the target because it is highly expressed on AML blasts and LSCs in most subtypes regardless of the patient's genetic characteristics and treatment course. Moreover, it is absent in the normal hematopoietic stem cells, granulocytes, naïve lymphocytes, and most normal nonhematopoietic tissues. Using a naïve human Fab phage display library, we isolated an anti-human TIM-3 antibody and designed a second-generation anti-TIM-3. Our anti-TIM-3 CAR T cells exhibit potent antileukemic activity against AML cell lines and primary AML blasts, and in the mouse models. More importantly, we demonstrate efficient killing of the primary LSCs directly isolated from the patients. Hence, eradication of the LSCs present in the MRD by anti-TIM-3 CAR T-cell therapy following the first-line treatment may improve the clinical outcomes of patients with AML.

Reactive Oxygen Species Drive Proliferation in Acute Myeloid Leukemia via the Glycolytic Regulator PFKFB3.

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a poor clinical outcome. Previously, we showed that overproduction of reactive oxygen species (ROS), arising from constitutive activation of NOX2 oxidase, occurs in >60% of patients with AML and that ROS production promotes proliferation of AML cells. We show here that the process most significantly affected by ROS overproduction is glycolysis. Whole metabolome analysis of 20 human primary AML showed that blasts generating high levels of ROS have increased glucose uptake and correspondingly increased glucose metabolism. In support of this, exogenous ROS increased glucose consumption while inhibition of NOX2 oxidase decreased glucose consumption. Mechanistically, ROS promoted uncoupling protein 2 (UCP2) protein expression and phosphorylation of AMPK, upregulating the expression of a key regulatory glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Overexpression of PFKFB3 promoted glucose uptake and cell proliferation, whereas downregulation of PFKFB3 strongly suppressed leukemia growth both in vitro and in vivo in the NSG model. These experiments provide direct evidence that oxidase-derived ROS promotes the growth of leukemia cells via the glycolytic regulator PFKFB3. Targeting PFKFB3 may therefore present a new mode of therapy for this disease with a poor outcome. SIGNIFICANCE: These findings show that ROS generated by NOX2 in AML cells promotes glycolysis by activating PFKFB3 and suggest PFKFB3 as a novel therapeutic target in AML.

The Mitochondrial Protease, Neurolysin (NLN), Regulates Respiratory Chain Complex and Supercomplex Formation and Is Necessary for AML Viability

Acute myeloid leukemia (AML) cells and stem cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation (OXPHOS). To identify new biological vulnerabilities in the mitochondrial proteome of AML cells, we conducted an shRNA screen and identified neurolysin (NLN), a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML has not been previously reported. To begin our investigation into the role of NLN in AML, we analyzed NLN gene expression in a database of 536 AML and 73 normal bone marrow samples. NLN was overexpressed in 41% of AML samples. Overexpression of NLN in primary AML cells compared to normal hematopoietic cells was confirmed by immunoblotting. To validate the results of the screen and to determine whether NLN is required for AML growth and viability, we knocked down NLN in the leukemia cell lines OCI-AML2, MV4-11, NB4, and TEX with shRNA. NLN knockdown reduced leukemia growth and viability by 50-70%. Moreover, knockdown of NLN in AML cells reduced the clonogenic growth of leukemic cells in vitro and the engraftment of AML cells into mouse marrow after five weeks by up to 80% and 85%, respectively. The mitochondrial function of NLN is largely unknown, so we identified NLN's mitochondrial protein interactors in T-REx HEK293 cells using proximity-dependent biotin labeling (BioID) coupled with mass spectrometry (MS). This screen identified 73 mitochondrial proteins that preferentially interacted with NLN and were enriched for functions including respiratory chain complex assembly, respiratory electron transport, and mitochondrion organization. Therefore, we assessed the effects of NLN knockdown on OXPHOS. NLN knockdown reduced basal and maximal oxygen consumption, but there were no changes in the levels of individual respiratory chain complex subunits. To understand how NLN influences OXPHOS, we examined the formation of respiratory chain supercomplexes (RCS). Respiratory chain complexes I, III, and IV assemble into higher order quaternary structures called RCS, which promote efficient oxidative metabolism. NLN knockdown significantly impaired RCS formation in T-REx HEK293, OCI-AML2, and NB4 cells, which was rescued by overexpressing wild-type shRNA-resistant NLN. RCS have not been previously studied in leukemia. Therefore, we analyzed their levels in primary AML patient samples and normal hematopoietic cells. RCS assembly was increased in a subset of AML patient samples and positively correlated with NLN protein expression (R2 = 0.83, p < 0.05), suggesting that NLN mediates RCS assembly in AML. To investigate how NLN may be regulating RCS assembly, we analyzed our BioID results to identify NLN interactors that are known regulators of supercomplex formation. Among the top interactors was the known RCS regulator, LETM1. Knockdown of NLN in AML cells impaired LETM1 assembly. Of note, knockdown of LETM1 also reduced growth and oxygen consumption of AML cells. As a chemical approach to evaluate the effects of NLN inhibition on AML cells, we used the allosteric NLN inhibitor R2, (3-[(2S)-1-[(3R)-3-(2-Chlorophenyl)-2-(2-fluorophenyl)pyrazolidin-1-yl]-1-oxopropan-2-yl]-1-(adamantan-2-yl)urea), whose anti-cancer effects have not been previously reported. R2 reduced viability of AML cells, as well as two primary AML culture models, 8227 and 130578. R2 impaired RCS formation in OCI-AML2, NB4, 8227, and primary AML cells. Moreover, R2 reduced the CD34+CD38- stem cell enriched population in 8227 cells, reduced LETM1 complex assembly, and impaired OXPHOS in OCI-AML2 and 8227 cells. Finally, we assessed the effects of inhibiting NLN in mice engrafted with primary AML and normal hematopoietic cells in vivo. Treatment of mice with R2 reduced the leukemic burden in these mice without toxicity. Moreover, inhibiting NLN targeted the AML stem cells as evidenced by reduced engraftment in secondary experiments. In contrast, inhibiting NLN did not reduce the engraftment of normal hematopoietic cells. Collectively, these results demonstrate that inhibition of NLN preferentially targets AML cells and stem cells as compared to normal hematopoietic cells. In summary, we defined a novel role for NLN in RCS formation. We show that RCS are necessary for oxidative metabolism in AML and highlight NLN inhibition as a potential therapeutic strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding. Schimmer:Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Abstract 1131: IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that occurs disproportionately in the elderly population. First line therapy is a regimen of anthracycline and cytarabine treatment or, more recently, demethylating agents. However, elderly AML patients are unable to tolerate aggressive chemotherapy regimens and there is an urgent need for targeted, tolerable therapies. To identify candidate pathways for targeted treatments, a microarray on transcripts for inflammatory signaling was performed on 588 primary patient AML samples. Non-hierarchical clustering revealed that Toll-like receptor (TLR) signaling transcripts are overexpressed in myelomonocytic and monocytic AML subtypes (M4 and M5 AML subtypes, respectively). Myeloid differentiation primary response 88 (MYD88) and downstream IL-1 receptor-associated kinases (IRAKs) were found to be overexpressed in these AML subtypes. MYD88 is an adaptor molecule for IL-1 and most TLR mediated signaling. To determine whether MYD88-dependent signaling is required for leukemogenesis, we derived a CreERT2-LoxP inducible system to delete Myd88 in a murine leukemia model. Because M4 and M5 AML subtypes are enriched for mutations in the mixed lineage leukemia (MLL) gene and overexpressed TLRs, MYD88, and IRAK2, an MLL-AF9 expressing construct was utilized to transform murine progenitor cells into leukemia cells. To study whether Myd88-dependent signaling is required for in vitro colony formation of the leukemic cells, Myd88 knockout (Myd88-/-) MLL-AF9 leukemic cells were subjected to colony-forming assays. Compared to Myd88 wild-type (Myd88fl/fl and Myd88+/+) leukemia cells, reduced colony-forming capacity was observed in the Myd88-/- leukemia cells. Further, reduced proliferation was observed in Myd88-/- cells compared to Myd88+/+ cells. Cell surface staining indicates Myd88-/- cells exhibit a loss of CD117, a marker of myeloid stemness, and a gain of CD11b, a marker of myeloid differentiation, when compared to Myd88+/+ cells, indicating that Myd88 deletion correlates with MLL-AF9 leukemia cell partial differentiation. In vivo transplantation of Myd88-/- cells delays leukemia development compared to Myd88+/+ cells. To determine whether IRAK1, a downstream kinase of MYD88, is also required to prevent partial differentiation, an IRAK1 shRNA knockdown cell line was generated and assayed for partial differentiation. In IRAK1KD MLL-AF9 cells, no partial differentiation was detected. Similarly, application of an IRAK1/4 inhibitor to Myd88+/+ MLL-AF9 cells failed to induce partial differentiation, despite suppressing proliferation. This indicates that MYD88-dependent signaling has functions independent of IRAK1 signaling that prevent partial differentiation in MLL-AF9 leukemia cells. Thus, MYD88 and IRAK1 signaling appear to mediate divergent functions, and both MYD88 and IRAK1 function appear to be required for MLL-AF9 leukemia. Citation Format: Joseph M. Cannova, Wei Wei, Rafael Gutierrez, Peter Breslin, Jiwang Zhang. IRAK1 and MYD88 mediate divergent signaling functions in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1131.

Abstract 1787: COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive blood cancer with neoplastic infiltration of leukemic blasts in blood, bone marrow and viscera. Current treatment options have limited efficacy, and the 5-year survival rate is only 27%. CD3 bispecific antibodies that re-direct T cells towards the tumor have shown promising efficacy in hematological malignancies. The CD3/CD19 bispecific antibody blinatumomab has recently been approved by the FDA for the treatment of B-cell acute lymphocytic leukemia (ALL) (Kantarjian H et al (2017) NEJM). We evaluated COVA4231, a CD3/CD33 bispecific FynomAb, preclinically as therapeutic candidate for AML. CD33 is a cell surface receptor expressed on blasts of the majority of AML patients (Walter RB (2014) Expert Opin Ther Targets). COVA4231 was constructed by fusing the CD33-specific Fynomer D5 - a small (7 kDa) globular protein derived from the human Fyn SH3 domain with engineered affinity for CD33 - to a CD3-specific antibody with a novel silent IgG1 Fc. COVA4231 induced T cell activation and cytokine release in a CD33-dependent manner, and elicited potent T cell mediated cytotoxicity of CD33-expressing target cells in vitro with EC50 in the range of 4 - 29 pM (E:T cell ratio of 2:1, KG-1 or MOLM-13 as target cells). COVA4231 showed potent activity against 13 out of 15 primary AML blast samples ex vivo at E:T cell ratio of 1:1. The in vivo efficacy of COVA4231 was investigated in a subcutaneous HL-60 leukemia xenograft model in NSG mice in the presence of human T cells. COVA4231 was highly active across a wide dose range (0.05 - 5 mg/kg) and prevented tumor outgrowth in all treated mice. COVA4231 demonstrated an IgG1-like pharmacokinetic profile in mice with a terminal half-life 15.8 days (total drug), providing the opportunity to avoid continuous intravenous infusion protocols required for established CD3 bispecific formats (e.g. (scFv)2). In conclusion, COVA4231 is a highly active therapeutic candidate in vitro, in vivo and ex vivo, has IgG-like pharmacokinetics and is a promising therapeutic candidate for further preclinical and clinical development. Citation Format: Kristina Klupsch, Vanessa Baeriswyl, Roland Scholz, Joana Dannenberg, Roger Santimaria, David Senn, Elena Kage, Adrian Zumsteg, Isabella Attinger-Toller, Ulrike von der Bey, Susann König-Friedrich, Fanny Dupuy, Wibke Lembke, Clara Albani, Severin Wendelspiess, Lucijana Dinkel, Chelsea J. Gudgeon, Roland B. Walter, Julian Bertschinger, Simon Brack. COVA4231, a potent CD3/CD33 bispecific FynomAb with IgG-like pharmacokinetics for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1787.

Abstract 1938: Store-operated calcium signaling is an effective therapeutic target in AML

Abstract Acute myeloid leukemia (AML) is the most prevalent adult leukemia characterized by genetic or mutational disruption of myeloid differentiation, growth arrest, and apoptosis. Standard “7 + 3” chemotherapy consisting of cytarabine in combination with an anthracycline such as daunorubicin has been used for more than four decades. However this course only results in 10 year disease free survival in 15% of patients age <60 years and 2% among those > 60 years. Several new successful therapies in AML are shown to work by promoting differentiation, and/or inducing apoptosis of the terminally differentiated cells. However these therapies, while promising, have the limitation of working in only a subset of AML with specific genetic profiles. The discovery of novel agents applicable to a broader patient population still represents a critical unmet need in this disease. Intracellular calcium is a common signaling molecule used in a variety of cellular processes including cell cycle progression. One of the major pathways which regulates calcium entry is store operated calcium entry (SOCE) mediated through calcium release-activated Ca2+ channels (CRAC), which respond to depleted calcium stores in ER lumen. This primarily occurs via interaction of ORAI1 (expressed on the plasma membrane) and STIM1 (expressed on the endoplasmic reticulum). Leukemic cells in particular often rely on the influx of calcium through CRAC for proliferation, therefore targeting these channels are a promising therapeutic option. In the current study, we explore the preclinical use of RP4010, a novel inhibitor of ORAI1 developed by Rhizen Pharmaceuticals, in AML. RP4010 is very effective at inhibiting proliferation of both AML cell lines and primary AML patient samples (IC50 values ranging from 0.2 - 1 μM using a tetrazolium-based colorimetric assay (MTS) assay). Importantly, RP4010 efficacy is not altered in the presence of stromal cell support. RP4010 also decreased colony growth in CFU assays. Finally, we evaluated the in vivo efficacy using an MV4-11 xenograft model. RP4010 treatment at 50 mg/kg once daily by oral gavage significantly prolonged survival compared to the vehicle control (p = 0.019). Furthermore, at the time of sacrifice, splenic tumor burden (percentage of human CD45 positive cells) was reduced in the mice treated with RP4010 compared to the vehicle group (p = 0.019). In conclusion, we provide here compelling evidence that inhibition of ORAI1 with RP4010 is a promising therapeutic strategy in AML. A Phase 1/1b study in R/R Non-Hodgkin Lymphoma (NHL) patients is currently underway in the US (ClinicalTrials.gov Identifier: NCT03119467), and our data would support expansion to other hematological malignancies as well, particularly AML. Senior authors J. C. B and E. H contributed equally to this work. Citation Format: Bridget Carmichael, Amy Lehman, Ola A. Elgamal, Shelley J. Orwick, Jean Truxall, Larry Beaver, Kumar V. Penmetsa, Srikant Viswanadha, John C. Byrd, Erin Hertlein. Store-operated calcium signaling is an effective therapeutic target in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1938.

A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402-13. ©2017 AACR.

MIF-Induced Stromal PKCβ/IL8 Is Essential in Human Acute Myeloid Leukemia

Acute myeloid leukemia (AML) cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo, indicating that tissue microenvironment plays a critical role in promoting AML cell survival. In vitro studies have shown that bone marrow mesenchymal stromal cells (BM-MSC) protect AML blasts from spontaneous and chemotherapy-induced apoptosis. Here, we report a novel interaction between AML blasts and BM-MSCs, which benefits AML proliferation and survival. We initially examined the cytokine profile in cultured human AML compared with AML cultured with BM-MSCs and found that macrophage migration inhibitory factor (MIF) was highly expressed by primary AML, and that IL8 was increased in AML/BM-MSC cocultures. Recombinant MIF increased IL8 expression in BM-MSCs via its receptor CD74. Moreover, the MIF inhibitor ISO-1 inhibited AML-induced IL8 expression by BM-MSCs as well as BM-MSC-induced AML survival. Protein kinase C β (PKCβ) regulated MIF-induced IL8 in BM-MSCs. Finally, targeted IL8 shRNA inhibited BM-MSC-induced AML survival. These results describe a novel, bidirectional, prosurvival mechanism between AML blasts and BM-MSCs. Furthermore, they provide biologic rationale for therapeutic strategies in AML targeting the microenvironment, specifically MIF and IL8. Cancer Res; 77(2); 303-11. ©2016 AACR.

Abstract 4712: Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML

Abstract Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Here, we describe INCB059872, a potent, selective and orally bioavailable inhibitor of LSD1 that achieves inhibitory activity through the formation of covalent FAD-adducts. INCB059872 inhibited cellular proliferation and induced cellular differentiation as measured by induction of CD86 and CD11b myeloid differentiation markers in a panel of human AML cell lines and primary human AML cells ex vivo. In vivo, pharmacodynamic (PD) assays confirmed the sustained induction of CD86 in human AML xenograft models, consistent with the mechanism of FAD-directed inhibition of LSD1. Oral administration of INCB059872 significantly inhibited tumor growth of human AML xenograft models as a single agent at doses exhibiting significant PD effects in vivo. Efficacy was further evaluated in the murine retroviral MLL-AF9 disseminated leukemia model that recapitulates hallmarks of human AML. INCB059872 significantly prolonged the median survival of MLL-AF9 expressing leukemic mice compared with vehicle treated animals. Mechanistic studies demonstrated that INCB059872 induced cell differentiation of murine blast cells, reduced blast colonies, and normalized clinical hematological parameters to those of non-leukemic mice. Notably, in both human AML xenografts and the murine MLL-AF9 leukemic model, maximal efficacy could be achieved with both daily (QD) and alternative-day (QoD) dosing regimens of INCB059872, consistent with the prolonged PD effects observed. Collectively, these studies demonstrate the key role that LSD1 activity can play in preventing leukemic cell differentiation, and support the therapeutic potential of INCB059872 in the treatment of human AML. Citation Format: Sang Hyun Lee, Matthew Stubbs, Xuesong Mike Liu, Melody Diamond, Valerie Dostalik, Min Ye, Yvonne Lo, Margaret Favata, Gengjie Yang, Karen Gallagher, Lynn Leffet, Chunhong He, Liangxing Wu, Alexander Margulis, Maryanne Covington, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Bruce Ruggeri, Peggy Scherle. Discovery of INCB059872, a novel FAD-directed LSD1 inhibitor that is effective in preclinical models of human and murine AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4712.

Abstract 4696: The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia

Abstract Acute myeloid leukemia (AML) is a disease characterized by the expansion of a hematopoietic stem cell like population caused in part by a block of myeloid differentiation. In AML, an altered epigenetic landscape, often arising from genetic lesions in epigenetic regulators, enforces an oncogenic expression profile and suppresses myeloid differentiation. Lysine specific demethylase 1 (LSD1) catalyzes the demethylation of lysine 4 and 9 of histone H3 through an FAD-dependent redox process. Aberrant LSD1 activity has been proposed to maintain oncogenic programs and prevent differentiation of multiple subtypes of AML. Intriguingly, recent studies indicate that LSD1 inhibition can reactivate an all-trans retinoic acid (ATRA)-dependent differentiation program in non-acute promyelocytic leukemia (APL) hematologic malignancies, a genetically heterogeneous group of blood cancers that normally respond poorly to ATRA therapy. In this study, we assessed the in vitro and in vivo effects of combining ATRA with INCB059872, a potent and selective FAD-directed LSD1 inhibitor, in non-APL AML models. As a single agent, INCB059872 induced differentiation of AML cells and when combined with ATRA, synergistically promoted differentiation as indicated by induction of CD86 and CD11b expression. In addition, the combination of INCB059872 and ATRA increased apoptosis in a panel of non-APL AML cell lines. Similarly, the combination significantly increased the fraction of CD86+CD11b+ cells and reduced cell viability in a panel of primary AML cells ex vivo. These effects in both human AML cell lines and human primary AML cells were observed across distinct FAB subtypes and genetic mutation profiles. Microarray profiling coupled with bioinformatic analysis of MV-4-11 cells demonstrated that the number of regulated genes related to differentiation and apoptotic pathways was markedly elevated in cells treated with the combination of LSD1 inhibition and ATRA relative to single agents. The synergistic increase in levels of myeloid lineage transcription factors GFI1, PU.1, CEBP and a decrease in levels of the oncogene c-MYC in the combination groups were validated by q-RT-PCR and western blot analyses. In vivo, the combination of INCB059872 and ATRA enhanced CD86 and CD11b induction and reduced tumor growth in the THP-1 xenograft model of AML compared with monotherapy. Similarly, oral administration of INCB059872 and ATRA in PDX mouse models markedly increased levels of CD11b+ cells in bone marrow. Collectively, these data underscore the synergy that can exist between LSD1 inhibition and retinoic acid receptor agonism, and provide a scientific rationale for the clinical evaluation of INCB059872 and ATRA in non-APL AML patients. Citation Format: Min Ye, Mike Liu, Jin Lu, Yvonne Lo, Margaret Favata, Gengjie Yang, Melody Diamond, Valerie Dostalik, Paul Waeltz, Melissa Ann Fischer, Chunhong He, Liangxing Wu, Xiaochuan Shan, Hong Chang, Maryanne Covington, Yanlong Li, Tim Burn, Richard Wynn, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle, Michael Savona, Martin Carroll, Bruce Ruggeri, Sang Hyun Lee. The LSD1 inhibitor INCB059872 is synergistic with ATRA in models of non-APL acute myelogenous leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4696.

Abstract LB-A15: IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe

Abstract Acute myeloid leukemia (AML) is a highly aggressive disease with a high mortality rate that encompasses several genetically and clinically diverse hematological malignancies characterized by clonal expansion of transformed stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of <12% for those aged >65 years. Therefore, novel, highly effective therapeutics are needed for this population. Targeting bioenergetic susceptibilities is an exciting area of oncology therapeutics that is potentially applicable in AML. Our group and others have shown that AML blasts depend significantly on mitochondrial oxidative phosphorylation to meet their energy and biomass production demands. Through an extensive medicinal chemistry campaign IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain with excellent PK and a suitable overall profile. In AML cell lines and primary AML blasts treated ex vivo, we observe a robust decrease in proliferation and a concomitant increase in apoptosis with EC50 values of less than 10 nM. Response to IACS-10759 in AML cells was associated with induction of a metabolic catastrophe that negatively impacted the cells' ability to sustain energy homeostasis, amino acid biosynthesis, and nucleotide production. In a primary AML patient derived xenograft model from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 (QDx5/week) treatment at 10 mg/kg extended the median survival by greater than 2-fold. Inhibition of OXPHOS by IACS-10759 was confirmed in AML cell lines and PDX models by a decrease in oxygen consumption and significant changes in gene and protein expression, non-essential amino acids and nucleotides. Due to the robust response in AML cell lines, primary AML samples ex vivo, and in vivo efficacy in primary AML PDX models, IACS-10759 has been advanced through IND enabling studies with first-in-human studies targeted for the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Maria Alimova, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Sonal, Jay Theroff, Quanyun Xu, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A15.

Intensive Chemotherapy in Patients Aged 70 Years or Older Newly Diagnosed With Acute Myeloid Leukemia

Acute myeloid leukemia (AML) represents a major therapeutic challenge in the elderly. Because of the high treatment-related mortality and poor overall outcomes of remission induction therapy, many older patients are not considered candidates for intensive chemotherapy. The current study evaluated prognostic factors for achievement of complete remission (CR) in newly diagnosed elderly AML patients who were treated with initial intensive chemotherapy. The study included 62 newly diagnosed AML patients ≥70 years who were treated with intensive chemotherapy. The overall response rate (CR and CRp) was 56%. Patients with favorable or intermediate cytogenetics (p = 0.0036) as well as those with primary AML (p = 0.0212) had a higher response rate. The median overall survival for all patients was 6.85 months (95% CI 3.7–13.5 months). The median overall survival for patients achieving remission after intensive induction chemotherapy was significantly higher than those who did not respond to therapy (20.4 months vs. 3.5 months, p < 0.001). The all-cause 4-week mortality rate was 11%, and the all-cause 8-week mortality rate was 17.7%. A subgroup of elderly patients may benefit more from initial intensive induction chemotherapy, specifically those patients with performance status able to tolerate induction chemotherapy and favorable cytogenetic status. However, despite high rates of initial CR, relapse rates are still high, suggesting that alternative strategies of postremission therapy are warranted.

CD99 Is a Therapeutic Target on Disease Initiating Stem Cells in Acute Myeloid Leukemia and the Myelodysplastic Syndromes

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) are initiated and sustained by self-renewing malignant stem cells. Any curative therapy for AML or MDS must eliminate leukemic stem cells (LSCs) or MDS hematopoietic stem cells (HSCs), respectively, as these are likely the only self-renewing disease cells. Through transcriptional profiling and flow cytometric validation, we previously identified CD99 as a cell surface marker highly expressed on MDS HSCs (85%, n=63). Assessment of 78 paired diagnosis/relapse AML specimens revealed that CD99 is also frequently overexpressed in AML at diagnosis (86%) and relapse (92%). Consistent with CD99’s potential as an LSC marker, we showed that the LSC enriched CD34+CD38- fraction of AML consistently expressed higher levels of CD99 compared with more differentiated blasts (p&lt;0.0001). We also found that CD99 expression was higher on leukemic blasts at relapse as compared with diagnosis (p=0.007). To test whether CD99 expression enriches for functional LSCs, we transplanted the top and bottom 10% of CD99 expressing blasts within the LSC enriched CD34+CD38-CD90-CD45RA+ fraction of a CD34-positive primary AML specimen into sublethally irradiated NOD/SCID/IL-2Rgc-null (NSG) mice, observing that leukemia-initiating cell (L-IC) activity was restricted to the top 10% of CD99 expressors (L-IC frequency 1 in 24,401). Similar results were obtained utilizing a CD34-negative AML (L-IC frequency 1 in 105,688 in the top 10% of CD99 expressors), suggesting that CD99 can enrich for functional LSCs independent of CD34-expression status. CD99 is the first LSC marker reported to selectively enrich for LSCs, as we found other previously identified LSC markers (CD47, TIM3, CD44, and CD123) to be expressed at equal or decreased levels on LSC-enriched fractions compared to more differentiated blasts. Thus, high CD99 expression provides a novel method to purify LSCs, allowing for more refined studies of this critical cell type. As therapeutically targeting CD99 with mAbs has been proposed in Ewing’s sarcoma and B cell acute lymphoblastic leukemia, we tested the ability of anti-CD99 mAbs to induce cell death in AML and MDS. Anti-CD99 mAbs (clones H036-1.1 and 12E7) were cytotoxic to primary AML blasts (n=5) and MDS CD34+ cells (n=2), as well as 17 myeloid leukemia cell lines in vitro. Normal HSCs and human umbilical vein endothelial cells express low and intermediate levels of CD99, respectively, but anti-CD99 mAbs were minimally toxic to these normal cell types, indicating a potentially large therapeutic window. Ex vivo incubation of primary AML blasts previously demonstrated to engraft NSG mice with H036-1.1 for 45 minutes prior to xenotransplantation completely abolished their engraftment. To rule out an effect on initial homing and lodging of LSCs, and to determine whether anti-CD99 mAbs can eradicate LSCs in vivo, NSG mice were transplanted with primary AML cells, allowed to engraft for two weeks, and then treated with 15 μg H036-1.1 or isotype control. One treatment with H036-1.1 was sufficient to completely abolish engraftment as measured in the BM five months post-treatment. To determine the mechanism of anti-CD99 mAb induced cell death, we assessed the effect of anti-CD99 mAbs on Src-family kinase (SFK) activation. We demonstrated that CD99 negatively regulates SFK activation in AML, and that cytotoxic anti-CD99 mAbs induce rapid and robust SFK activation. To determine whether SFK activation is sufficient to induce cell death, we generated a constitutively active c-Src mutant (Y530F) and overexpressed it in anti-CD99 mAb “sensitive” and “resistant” cell lines (MOLM13 and K562, respectively). While MOLM13 cells showed a marked decrease in cell growth, increased apoptosis, and G0 cell cycle arrest, K562 cells demonstrated only a modest decrease in growth and S-phase entry, with no G0 arrest or increase in apoptosis. We thus propose that anti-CD99 mAbs induce apoptosis by inducing dysregulated SFK activation and oncogenic stress. Taken together, our studies establish CD99 as a novel stem cell marker in AML and MDS, and as a promising therapeutic target in two diseases in need of novel therapies with validated activity against disease-initiating cells. Cytotoxic anti-CD99 mAbs appear to exert their effects by activating SFKs, revealing a novel therapeutic vulnerability in AML and MDS that has potential to be exploited using other modulators of this pathway. Disclosures No relevant conflicts of interest to declare.