Blast Phase Chronic Myeloid Leukemia Research Articles

NUP98-HOXA9-Initiates Molecular and Biologic Features of Disease Progression in a Humanized Model of CML

Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which >97%, >96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+. In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these. RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5' HOXA genes, 3' HOXB genes, PBX3, MEIS1,ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP,PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200). This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value <0.05). Finally, we identified super-enhancers in 12 genes among which were PBX3, ANGPT1, MBNL1 and PRKACB. Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype. Disclosures Turhan: novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.

Pro-Active Dasatinib Dose Reduction Based on Trough Levels May Minimise Toxicity and Preserve Efficacy - Interim Analysis of the ALLG CML 12 Direct Study

Pro-Active Dasatinib Dose Reduction Based on Trough Levels May Minimise Toxicity and Preserve Efficacy - Interim Analysis of the ALLG CML 12 Direct Study

Phase 2 Study of Bosutinib in Japanese Patients with Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Phase 2 Study of Bosutinib in Japanese Patients with Newly Diagnosed Chronic Phase Chronic Myeloid Leukemia

Ponatinib Treatment in CML and Ph+ ALL Patients: Real-Life Data from the Czech Registries

Patients with newly diagnosed chronic myeloid leukemia (CML) frequently receive imatinib. Although initial response rates are high, imatinib fails in up to 40% of patients because of disease resistance, frequently because of BCR-ABL kinase domain mutations, or side effects. Patients who discontinue imatinib may have a response to second-generation tyrosine kinase inhibitors (TKIs). Ponatinib (PON) is a potent oral TKI active against unmutated and mutated BCR-ABL kinase. PON is indicated also in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) patients. Clinical activity of PON was confirmed in the phase II PACE trial, however lack of real world data is evident. The aim of this non-interventional study was to analyze data on PON treatment and its efficacy in the Czech patients with CML / Ph+ ALL. The study was designed as a one-off retrospective data collection from 3 national registry databases INFINITY, CAMELIA and DATOOL ALL in the period 2014-2018. In total, the study comprised 27 patients treated with PON at 7 centers; 16 (59.3%) patients were treated for chronic phase (CP) CML, 4 (14.8%) patients for accelerated- or blast-phase (AP/BP) CML and 7 (25.9%) patients for Ph+ ALL. The 16 CP CML patients (68.8% males) had median age at the start of PON treatment 59.7 years (range 28.6-81.4), were heavily pretreated (75% with ≥ 3 TKIs) with median time from diagnosis to start of PON treatment 4.8 (0.2-16.8) years; 37.5% of them had mutations (18.8% with T315l), 75.0% had comorbidities. The 11 AP/BP CML / Ph+ ALL patients (54.5% males) were younger with median age 56.6 (31.2-74.7) years, were less pretreated (36.4% with ≥ 3 TKIs) with a shorter time from diagnosis to start of PON treatment 2.0 (0.4-22.0) years; almost twice as many (72.7%) of them had mutations (54.5% with T315l), 63.6% had comorbidities. The most common reason for switching to ponatinib was hematologic resistance (37.5% of CP CML and 54.5% of AP/BP CML / Ph+ ALL patients). The other most frequent indications were cytogenetic resistance, non-hematologic and hematologic intolerance (18.8%, 12.5% and 12.5% of CP CML patients and 9.1%, 9.1% and 0% of AP/BP CML / Ph+ ALL patients, respectively). Interestingly, starting dose of PON was 45 mg/day as recommended in the product SmPC only in of about half of the patients (43.8% of CP CML and 54.5% of AP/BP CML / Ph+ ALL patients). Median treatment duration was 16.1 (0.8-49.9) months in CP CML patients and only 2.9 (0.2-36.1) months in AP/BP CML / Ph+ ALL patients. Early (in the first 3 months) and late termination of the treatment occurred in 12.5% and 31.3% of CP CML and 54.5% and 27.3% of AP/BP CML / Ph+ ALL patients, respectively. Disease progression was the major reason for treatment termination (50%). In terms of safety, only 1 patient discontinued therapy due to congestive heart failure, and 1 due to vascular adverse event although more than half of the patients had cardiovascular comorbidities and history of cardiovascular disease. PON efficacy was evaluated in 14 CP CML patients and 5 AP/BP CML / Ph+ ALL who were treated beyond 3 months (Figure 1). More than half of CP CML patients achieved MMR (57.1%) and 40% of AP/BP CML / Ph+ ALL achieved undetectable disease. Estimated percentage of CHR, CCyR and MMR in CP CML patients after one year of treatment was 85.7%, 50% and 50%, respectively. Nevertheless, 5 (35.7%), 2 (14.3%) and 1 (7.1%) patients had CHR, CCyR and MMR at start of PON treatment, respectively. In AP/BP CML / Ph+ ALL patients, estimated percentage of CR and CMR after one year was 100% and 40%, respectively. However, 2 patients (20%) had CR at the start of PON treatment. Despite limited number of patients, our analysis confirmed PON efficacy in real-life setting with a significant proportion of heavily pre-treated patients achieving durable molecular responses in both CP and AP/BP CML / Ph+ ALL groups. Our data are comparable to the PACE trial results. This study partially fills the gap in RWE data and significantly contributes to the evaluation of real-life clinical practice in rare disease area. Figure 1. Cumulative incidence of responses on ponatinib treatment in A) CP CML (N=14) and B) AP/BP CML / Ph+ ALL (N=5) patients that continued ponatinib treatment beyond 3 months. Figure 1 Disclosures Žáčková: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; Angelini: Consultancy; Incyte: Consultancy. Kellnerová:Angelini Pharma: Employment.

Real-Life Outcomes of Ponatinib Treatment in Patients with Chronic Myeloid Leukemia (CML) and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL): Data from a Nationwide Belgian Registry

Real-Life Outcomes of Ponatinib Treatment in Patients with Chronic Myeloid Leukemia (CML) and Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL): Data from a Nationwide Belgian Registry

Accumulation of DNA Damage and Alteration of the DNA Damage Response in Chronic Myeloid Leukemia

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinases Exerts Synergistic Effect Against Chronic Myeloid Leukemia Cells

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of BCR-ABL1 tyrosine kinase - positive chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" the disease in majority of patients because CML-CP cells are elusive targets even for the most advanced therapies employing second and third generation of TKIs. Therefore, new treatment modalities are necessary to improve therapeutic outcomes. We showed before that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. The aim of the study was to test whether simultaneous inhibition of signaling pathways activated by BCR-ABL1 and PAK kinases may improve the treatment of CML. Special attention was focused on PAK1 and PAK2, which are expressed in hematopoietic cells and can play an important role in the promotion of CML cells proliferation and survival. PAK kinases were targeted by small molecule inhibitor IPA-3 (inhibitor of PAK1) and shRNA construct for PAK2, BCR-ABL1 kinase was inhibited by imatinib. The studies were carried out using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed CML-CP patients and healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) BCR-ABL1-transformed 32Dcl3 cell line cells. We show here that inhibition of PAK1 or/and PAK2 kinases activity enhanced the effect of IM against CML cells without affecting normal counterparts. We observed that the combined use of IM with IPA-3 increased growth inhibition and apoptosis of leukemia cells. To evaluate the type of drugs interaction median effect analysis method was used. The studies revealed that the type and strength of drug interaction depend on drug concentration. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Altogether, we postulate that BCR-ABL1 kinase inhibitor should be combined with PAK1/2 inhibitor to facilitate eradication of CML cells. Disclosures No relevant conflicts of interest to declare.

Morphine activates blast-phase chronic myeloid leukemia cells and alleviates the effects of tyrosine kinase inhibitors

Morphine is an opioid analgesic drug routinely used in the postoperative period for pain management in cancer patients. In this work, we analyzed the effects of morphine on leukemia cells at all stages of development and addressed its underlying mechanism. We showed that clinically relevant concentrations of morphine promoted growth without affecting survival in blast phase-chronic myeloid leukemia (BP-CML) K562 and LAMA84 cells. In addition, morphine alleviated the anti-proliferative and pro-apoptotic effects of BCR-ABL tyrosine kinase inhibitor (TKI) in BP-CML cells. We further found that morphine increased colony formation and replating capacity of CD34 stem/progenitor derived from BP-CML patients. In addition, morphine alleviated the inhibitory effects of BCR-ABL TKIs in cell survival, colony formation and replating capacity in BP-CML CD34 + stem/progenitor cells. Mechanistic investigations demonstrated that morphine specifically activated Wnt signaling via increasing β-catenin activity and Wnt target genes transcription in K562 and CD34 + stem/progenitor cells. The effects of morphine in BP-CML were abolished by Wnt inhibitor LGK-974 or XAV939, which further confirmed that morphine protected BP-CML cells from BCR-ABL TKIs-induced toxicity via activating Wnt/β-catenin signaling. Our work demonstrated the novel role of morphine on leukemia cells. The activation of Wnt/β-catenin by morphine may provide a new guide in the clinical use of morphine, particularly in patients with Wnt-driven cancers.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinase Exerts Synergistic Effect against Chronic Myeloid Leukemia Cells.

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia in the chronic phase (CML-CP). However, it is unlikely that they can completely “cure” the disease. This might be because some subpopulations of CML-CP cells such as stem and progenitor cells are resistant to chemotherapy, even to the new generation of TKIs. Therefore, it is important to look for new methods of treatment to improve therapeutic outcomes. Previously, we have shown that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. In this study, we aimed to determine if simultaneous inhibition of BCR-ABL1 oncogenic tyrosine kinase and PAK1/2 serine/threonine kinase exert better anti-CML effect than that of individual treatments. PAK1 was inhibited by small-molecule inhibitor IPA-3 (p21-activated kinase inhibitor III), PAK2 was downregulated by specific short hairpin RNA (shRNA), and BCR-ABL1 tyrosine kinase was inhibited by imatinib (IM). The studies were conducted by using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed patients with CML-CP and from healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) from BCR-ABL1-transformed 32Dcl3 cell line. Herein, we show that inhibition of the activity of PAK1 and/or PAK2 enhanced the effect of IM against CML cells without affecting the normal cells. We observed that the combined use of IM with IPA-3 increased the inhibition of growth and apoptosis of leukemia cells. To evaluate the type of interaction between the two drugs, we performed median effect analysis. According to our results, the type and strength of drug interaction depend on the concentration of the drugs tested. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Based on our results, we hypothesize that IM, a BCR-ABL1 tyrosine kinase inhibitor, combined with a PAK1/2 inhibitor facilitates eradication of CML-CP cells.

Heterogeneous BCR-ABL1 signal patterns identified by fluorescence in situ hybridization are associated with leukemic clonal evolution and poorer prognosis in BCR-ABL1 positive leukemia

BackgroundAlthough extensive use of tyrosine kinase inhibitors has resulted in high and durable response rate and prolonged survival time in patients with BCR-ABL1 positive chronic myeloid leukemia (CML) and acute leukemia, relapse and drug resistance still remain big challenges for clinicians. Monitoring the expression of BCR-ABL1 fusion gene and identifying ABL kinase mutations are effective means to predict disease relapse and resistance. However, the prognostic impact of BCR-ABL1 signal patterns detected by fluorescence in situ hybridization (FISH) remains largely unaddressed.MethodsBCR-ABL1 signal patterns were analyzed using FISH in 243 CML-chronic phase (CML-CP), 17 CML-blast phase (CML-BP) and 52 BCR-ABL1 positive acute lymphoblastic leukemia (ALL) patients.ResultsThe patterns of BCR-ABL1 signals presented complexity and diversity. A total of 12 BCR-ABL1 signals were observed in this cohort, including 1R1G2F, 1R1G1F, 2R1G1F, 1R2G1F, 2R2G1F, 1R2G2F, 1R1G3F, 1G3F, 2G3F, 1G4F, 1R1G4F and 1R4F. Complex BCR-ABL1 signal patterns (≥ two types of signal patterns) were observed in 52.9% (n = 9) of the CML-BP patients, followed by 30.8% (n = 16) of the ALL patients and only 2.1% (n = 5) of the CML-CP patients. More importantly, five clonal evolution patterns related to disease progression and relapse were observed, and patients with complex BCR-ABL1 signal patterns had a poorer overall survival (OS) time compared with those with single patterns (5.0 vs.15.0 months, p = 0.006).ConclusionsOur data showed that complex BCR-ABL1 signal patterns were associated with leukemic clonal evolution and poorer prognosis in BCR-ABL1 positive leukemia. Monitoring BCR-ABL1 signal patterns might be an effective means to provide prognostic guidance and treatment choices for these patients.

B-Lymphoid Blast Phase of Chronic Myeloid Leukemia: A Case Report and Review of the Literature

Abstract Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder characterized by a reciprocal translocation, t(9;22)(q34.1;q11.2). This leads to fusion of the BCR and ABL1 genes, encoding an active tyrosine kinase that causes unregulated proliferation of the myeloid lineage. The BCR/ABL1 fusion protein is found not only in CML, but also in a subset of de novo B-lymphoblastic leukemia (B-LL). However, the fusion protein in CML is characteristically the slightly longer p210 variant, whereas the p190 variant is more frequently found in B-LL. Without treatment, CML will progress to accelerated and/or blast phase (BP). Disease progression is often characterized by accumulation of additional chromosomal abnormalities. The development of tyrosine kinase inhibitor (TKI) therapy that targets BCR/ABL1 has revolutionized treatment of CML and vastly improved outcomes, although the disease can still progress despite TKI therapy. Blast phase most commonly manifests as myeloid BP; however, up to 30% of BP presents as lymphoid BP (LBP), typically of the B-cell lineage. The B-lymphoblasts of LBP have a phenotype indistinguishable from that of de novo B-LL. However, LBP typically carries the p210 BCR/ABL transcript and may show distinct chromosomal anomalies, including loss of chromosome 9p. The prognosis for CML-BP is poor, although survival has improved with TKI therapy and stem cell transplant, and LBP has been associated with superior survival compared with myeloid BP. Here we present a case of CML in B-lymphoid BP and review the current literature.

Lysosome Inhibition by Mefloquine Preferentially Enhances the Cytotoxic Effects of Tyrosine Kinase Inhibitors in Blast Phase Chronic Myeloid Leukemia

Despite the efficacy of BCR-ABL tyrosine kinase inhibitors (TKIs) in chronic phase-chronic myeloid leukemia, the management of blast phase-chronic myeloid leukemia (BP-CML) remains a challenge. Therefore, there is an urgent need to identify alternative agents that act synergistically with BCR-ABL TKIs in BP-CML. Our results show that the anti-malarial agent, mefloquine augments the efficacy of TKIs in CML cell lines and primary CML cells in vitro, including those with the T315I mutation. This effect is selective as mefloquine is more effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells derived from TKI-resistant BP-CML patients than normal cord blood (CB) CD34+ stem/progenitor cells. Notably, the combination of mefloquine and TKIs at sublethal concentrations leads to synergistic effects in CML CD34+ cells, while sparing normal CB CD34+ cells. We further demonstrate that the initial action of mefloquine in CML cells is to increase lysosomal biogenesis and activation, followed by oxidative stress, lysosomal lipid damage and functional impairment. Taken together, our work elucidates that mefloquine selectively augments the effects of TKIs in CML stem/progenitor cells by inducing lysosomal dysfunction.

Abstract 3018: Mechanisms of tyrosine kinase inhibitor resistance in chronic myeloid leukemia

Abstract Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic disease. Despite improved survival, resistance is a clinical problem, and TKIs do not target the quiescent CML leukemic stem cell (LSC), meaning that patients must be treated for life at a high economic burden and sometimes with significant side effects. TKI resistance is frequently characterized by mutations in the BCR-ABL1 kinase domain, but they explain only ~50% of clinical TKI failure. The remaining patients have BCR-ABL1-independent resistance, defined as survival despite BCR-ABL1 inhibition. We have previously reported a gene expression signature predictive of TKI failure in CD34+ cells from chronic phase CML patients (McWeeney et al. 2010). This gene expression signature demonstrated significant overlap with signatures of blast phase CML (Zheng et al. 2006), suggesting that similar biological processes may be driving TKI resistance and disease progression. We expanded our analysis, and also found significant overlap between the expression profiles of TKI resistance and quiescent CML LSCs reported by Graham et al. (p=4x10-14) and Cramer Morales et al. (p=7x10-6). These data suggest that there is a core of genes whose expression is consistently associated with multiple scenarios of BCR-ABL1-independent resistance. Our previous work has shown that BCR-ABL1-independent resistance is largely driven by STAT3, and that targeting STAT3 in combination with TKIs restores sensitivity in TKI-resistant CML stem and progenitor cells (Eiring et al. Leukemia 2015). Unexpectedly, gene set enrichment analysis revealed that our gene expression signature predictive of TKI failure (McWeeney et al. 2010) does not reveal a STAT3 transcriptional signature. Similarly, RNA sequencing data on TKI-resistant K562-R cells, which are resistant to TKIs but lack BCR-ABL1 kinase domain mutations, revealed that, while TKI resistance was not associated with a STAT3 transcriptional signature (p=1.0), it was correlated with a signature reminiscent of TNFα signaling via NFκB (p=0.024). Nucleocytoplasmic fractionation revealed higher levels of total- and phospho-NFκB in the nucleus of K562-R versus K562-S cells, and in CD34+progenitors from TKI-resistant CML patients (n=3) compared to TKI responders (n=2) and normal individuals (n=2). These data indicate that NFκB may be driving the gene expression signature associated with BCR-ABL1-independent resistance, and suggest non-canonical functions for STAT3 that go beyond its traditional role as a transcription factor. Citation Format: Anna M. Eiring, Rebecca Ellwood, Carme Ripoll Fiol, Robert K. Hills, Georgios Nteliopoulos, Alistair Reid, Dragana Milojkovic, Jane Apperley, Jamshid Sorouri-Khorashad. Mechanisms of tyrosine kinase inhibitor resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3018.

PF189 APPROPRIATE INTERVAL OF MONITORING FOR DETECTION OF MOLECULAR RELAPSE AFTER STOP OF TKI IN PH+ALL IN PH+ ALL CALCULATING BY MRD KINETICS

Please indicate where the abstract has been published before: Blood 2018 132:5291; doi: https://doi.org/10.1182/blood‐2018–99–112293Background:Usually TKI is stopped in patients with Ph+ leukemia (CML/ALL) who have sustained a deep molecular response. The MRD monitoring interval and threshold of restarting TKI is mostly established in CML. In Ph+ALL patients, prophylactic/maintenance administrations of TKI are used during post hematopoietic stem cell transplantation (HSCT) as well as post chemotherapy. Unlike CML, no established selection of patients who can stop TKI. In addition, after TKI stopped, there is no consensus about how often to monitor MRD and threshold of restart for TKI.Aims:In the current study, we tried to determine the optimal frequency of MRD monitor after the cessation of TKI in Ph+ALL using a mathematical model.Methods:Mimicking CML, we defined BCR‐ABL/ABL ratio of 0.1% as“MR3” and undetectable levels of BCR‐ABL transcript as “MR5”. We estimate that patients with MR3 and MR5 have 109 and 107 Ph+ cells in the body, respectively. In this perspective, we defined “MR3” and “MR5” as “optimal intervention threshold” and “detection threshold”. From the literatures, we determined that growth rate of leukemia is distributed between 1%/day and 100%/day (doubling time of leukemic cells is 1 day). Also, we assumed that single cell have a relapse potency and the growth rate is constant during observation in each patient.Results:Table shows the interval from single leukemia cell to MR5 leukemia burden (1 x 107 cells) and the interval from MR5 to MR3 (X100) according to growth rate. Figure is a graph of Table. We calculated using most extreme cases. In the highest growing speed (100%/day), if the number of Ph+ cells is just below detection level (MR5), it will take 6.6 days to reach MR3 (optimal intervention threshold). Also, if the number of Ph+ cells is only 1 cell, it will take 23.3 days to reach MR5. The other ultimate condition is that the number of leukemia cells is only one in the body and has the slowest growth rate (1%/day). In this case it will take 1620 days (4.4 years) to reach MR5. It was reported that the growth rate of Ph+ cells in the blastic phase of CML is approximately 8% (doubling time 9 days) (Branford. Blood. 2012;119:4264). Applying this value to our model, it will take 209 days to reach MR5 (detection threshold) from single Ph+ cell. If the number of leukemia cells is just below the detection level (MR5), it will take 60 days to reach MR3 (optimal intervention threshold). In this patient a 2 months interval can detect MRD before it reaches to MR3 (Success). If 1 Ph+ cell remains when TKI is stopped, it will reach the MRD detection level (MR5) in 269 days. If Ph+ cells does not appear until this time, this patient is supposed to be cured in this model. In the clinical setting, growth rate is unknown. Thus in the beginning, a highest growth rate of 100% is applied. (Figure) As mentioned above, monitoring should be performed every 6.6 days until 23.3 days, At this point, interval MRD monitoring can be prolonged to 23.3 days until 81.7 days. (Likewise interval of 81.7 days until 285.8 days and interval of 290.1 days until 1015 days)Summary/Conclusion:Taken together, soon after stop of TKI, frequent monitoring of MRD is needed. The interval can be prolonged with the passage of time. After allogeneic HSCT, the growth rate of leukemia may be down regulated to some extent according to the GVL effect. The current strategy may be applied to other leukemia in which MRD monitoring by PCR is established.image

Diagnostic challenges of de novo acute leukemia with positive BCR-ABL1 fusion: Case report and review of literature

A 32-year-old female was admitted because of an acute course of gum bleeding and fatigability. On complete blood cell counts, bicytopenia was evident. Morphologic evaluation of the peripheral blood smear and bone marrow materials revealed remarkable involvement by two separate blastic populations (myeloid/B-cell lymphoid). Karyotyping, fluorescence in situ hybridization, and molecular analysis showed positive Philadelphia chromosome and BCR-ABL1 (p210) fusion. chronic myeloid leukemia in blast phase (CML-BP) versus mixed phenotype acute leukemia was critically debated as to which the final diagnosis should be assigned.

BRD4 Degradation Is a Potent Approach to Block MYC Expression and to Overcome Multiple Forms of Stem Cell Resistance in Ph+ CML

Chronic myeloid leukemia (CML) is a hematopoietic stem cell neoplasm in which BCR-ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients, leukemic cells can be kept under control by BCR-ABL1 tyrosine kinase inhibitors (TKI). Nevertheless, resistance against one or more TKI may occur. Therefore, research is focusing on novel potential drug targets in CML. We have recently identified the epigenetic reader bromodomain-containing protein 4 (BRD4) as a new therapeutic target in leukemic stem cells (LSC) in acute myeloid leukemia. In the present study, we examine the expression of BRD4 and its downstream effector MYC in CML cells and asked whether BRD4 serves as a drug target in CML cells and whether BRD4-targeting drugs, including JQ1 and newly developed BRD4 degraders (dBET1 and dBET6) are able to overcome LSC resistance in CML. Primary CML cells were obtained from 22 patients with chronic phase (CP) CML and 3 with blast phase (BP) CML. As determined by qPCR and/or immunocytochemistry, the CML cell lines KU812 and K562 as well as primary CML cells expressed BRD4 and MYC. All three BRD4-targeting drugs (JQ1, dBET1 and dBET6) were found to decrease MYC expression in KU812 and K562 cells as assessed by Western blotting. In 3H-thymidine uptake experiments, JQ1 and dBET6 were found to inhibit the proliferation of KU812 in a dose-dependent manner (IC50, JQ1: 100-500 nM; dBET6: 50-100 nM) whereas dBET1 showed only little if any effects on growth of KU812 cells (IC50: 1-5 µM), and in K562 cells, only dBET6 was found to inhibit growth with a reasonable IC50 value (250-500 nM). Corresponding results were obtained when examining drug effects on survival of CML cell lines by Annexin-V/PI staining. All three BRD4-targeting drugs were found to inhibit proliferation of primary CP CML cells with varying IC50 values. As expected, growth-inhibitory effects of dBET6 were more pronounced (IC50: <100 nM) compared to effects seen with JQ1 and dBET1. dBET1 and dBET6 were also found to inhibit growth of primary CML cells obtained from patients with BP CML, whereas JQ1 was not effective. JQ1 also failed to suppress survival on CML CD34+/CD38− LSC. By contrast, dBET1 induced apoptosis in CML LSC at 1 µM and dBET6 induced apoptosis in CML LSC at 0.1 µM. dBET6 induced apoptosis in CML LSC obtained from patients with imatinib-sensitive CML as well as patients with imatinib-resistant CML harboring BCR-ABL1 T315I or BCR-ABL1 F317L. Finally, pre-incubation of CD34+ CP CML cells with dBET6 resulted in reduced leukemic engraftment in NSG mice exhibiting human membrane-bound stem cell factor, SCF [NSG-Tg(hu-mSCF)] 6 months after transplantation (engraftment with CD45+/CD33+/CD19−cells in control mice receiving DMSO-treated cells: 8.1±6.6% vs mice receiving dBET6-treated cells: 1.1±0.6%). To further explore the ability of dBET6 to interfere with LSC resistance in CML, we established a co-culture system mimicking LSC-niche interactions in the osteoblastic niche. In this model, co-culturing K562 cells, KU812 cells or primary CML LSC with the osteoblast-like osteosarcoma cell line CAL-72 resulted in resistance against nilotinib and ponatinib. In this culture system, JQ1 was found to partially restore TKI effects in K562 cells and completely restored TKI effects in KU812 cells. Interestingly, JQ1 was not able to restore TKI effects in primary CML LSC in these co-cultures. However, dBET6 was found to overcome niche cell-induced TKI-resistance of primary CML LSC. Finally, we were able to demonstrate that JQ1, dBET1 and dBET6 inhibit interferon-gamma-induced upregulation of PD-L1 expression in CML LSC. Together we show that BRD4 and MYC are potential new therapeutic drug targets in CML and that the BET-degrader dBET6 overcomes multiple forms of LSC resistance, including i) intrinsic resistance, ii) mutation-induced resistance, iii) niche induced resistance and iv) checkpoint-mediated resistance. Whether BRD4 degradation is also able to overcome TKI-resistance of BCR-ABL1+ LSC in vivo in patients with CML remains to be determined in clinical trials. DisclosuresHoermann:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria. Wolf:BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; AOP Orphan: Honoraria, Research Funding. Mayer:Amgen: Research Funding; Novartis: Research Funding. Zuber:Mirimus Inc.: Consultancy, Other: Shareholder; Boehringer Ingelheim GmbH & Co KG: Research Funding. Sperr:Novartis: Honoraria; Pfizer: Honoraria; Daiichi Sankyo: Honoraria. Valent:Pfizer: Honoraria; Incyte: Honoraria; Novartis: Honoraria.

RUNX1 Mutations Identify an Entity of Blast Phase Chronic Myeloid Leukemia (BP-CML) Patients with Distinct Phenotype, Transcriptional Profile and Drug Vulnerabilities

Introduction: Genetic alterations of RUNX1 gene (mutations and fusion genes) are common in hematological malignancies including AML, ALL, MDS, and MPN. Mutations of RUNX1 occur in 10% of newly diagnosed AML patients and associate with inferior prognosis. Similarly, point mutations in RUNX1 are frequent in BP-CML, but little is known about their role in BP patients.Methods: Bone marrow samples from four BP patients with RUNX1 mutations were collected from Helsinki University Hospital, and samples from four other BP patients without RUNX1 mutations were used as controls. Samples were analyzed by exome and RNA sequencing. For drug sensitivity and resistance testing (DSRT), a high-throughput platform comprising 195 approved and investigational oncology drugs was used. To further study the role of RUNX1 mutations in BP-CML, Baf3/BCR-ABL1+ RUNX1-/- knock-out and RUNX1-/mut heterozygous deletion mutant models were created using CRISPR-CAS9 technology. The effects of the mutations in the cell line models were characterized by flow cytometry, RNA sequencing, and DSRT analyses.Results: The median age of RUNX1-mutated BP patients was 47 years (range 36-56 years) and included 3 patients with myeloid-BP and 1 with lymphoid-BP phenotype. RUNX1-mutated patients tended to have higher mutational load and co-occurrence with mutations to the BCOR, EZH2 and PHF6 genes in agreement with the mutational profile of RUNX1-mutated AML. Signature analysis of RUNX1-mutated BP patients showed dominance of signature 1 (age-related), signature 6, and 15 (DNA mismatch repair) correlating with the control group signature profiles. In addition, the RUNX1 mutated group showed specific enrichment of signature 9, related to somatic hypermutation and AID/RAG axis activity, that was not observed in the control group. RNA sequencing revealed that 388 genes were significantly differentially expressed between RUNX1-mutated and control BP patients (139 upregulated, 249 downregulated). Interestingly, the antigen processing and presentation pathway was among the top upregulated pathways in RUNX1 mutated patients with overexpression of CIITA and HLA-DR genes. Stem cell differentiation, complement, and hemostasis pathways were enriched in the downregulated gene set. Lymphoid-specific transcription factors (DNTT, PAX5 and VPERB1) in addition to CD19 and CD25 were significantly upregulated in the RUNX1-mutated group. DSRT data showed greater sensitivity to glucocorticoids, MEK-, mTOR- and VEGFR-inhibitors in RUNX1-mutated patients compared to the control group. Data from RUNX1-/- and RUNX1-/mut cell line models confirmed the induced expression of CD19 surface marker with RUNX1 mutation. DSRT results showed increased activity of MEK inhibitor and mTOR inhibitors in RUNX1-/- knock-out and RUNX1-/mut cell lines.Conclusions:RUNX1 mutations in BP-CML patients associate with distinct phenotypic and transcriptional features. DSRT results together with transcriptional data support the potential benefits of glucocorticoids and MEK and mTOR inhibitors as well as immunotherapy targeting CD19 and CD25 in this group of BP-CML patients. DisclosuresKankainen:Medix Biochemica: Consultancy. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding; Celgene: Honoraria.

34-Year Single Center Observational Review of Ex-Vivo T-Cell Depleted Allogeneic Hematopoietic Stem Cell Transplants for Chronic Myeloid Leukemia

Introduction:Ex-Vivo T-Cell Depletion (EX-TCD) has been shown, in multiple cohorts, to markedly decrease the incidence of graft-versus-host-disease (GvHD). This conditioning regime however, has been associated with high incidence of disease relapse. This single center, observational 34 year study, demonstrates outcomes of patients who underwent EX-TCD Allogeneic Hematopoietic Stem Cell Transplant (AHSCT) for Chronic Myeloid Leukemia (CML).The total cohort consisted of 62 patients; all underwent EX-TCD AHSCT between 1984 and 1988. 58% (n=36) of the total cohort relapsed and 53% of relapsed patients (n=19) went on to have a second AHSCT (non-T-cell depleted) or donor lymphocyte infusion (DLI) with the remaining 47% (n=17) receiving conservative management. Given these differences in management, we used a competing risk model to adjust for the second AHCST or DLI on survival outcomes (using a second AHSCT or DLI as a competing risk).The aim of this study was to determine from this cohort, which factors were associated with overall survival and survival following disease relapse.Methods:Within the relapsed cohort, univariate and multivariate standard Cox proportional hazard regression models were used to analyse various factors, which were hypothesized to be associated with survival. These included; age at relapse, gender and relapse grade. Competing risk model was used to analyse the contribution of a second AHSCT or DLI to survival.Results:The cohort underwent EX-TCD AHSCT for CML between 1984 and 1988. Age range of recipients was 15 to 53 years and all were sibling donors. At the 34 year follow-up mark, there was no difference in overall survival between those who relapsed (and received a second AHSCT or DLI) and those patients who did not relapse (p-value 0.86). Of the 26 % (n=16) of patients who remain alive, 50% (n=8) relapsed and received a second AHSCT or DLI. Most recent BCR-ABL polymerase chain reaction (PCR) confirm all patients remain in molecular remission (MR4 or below).For the whole cohort, relapse was not affected by; donor gender, recipient age, CMV status, GvHD grade, duration of disease prior to AHSCT, Sokal score at diagnosis or duration to relapse following AHSCT. No parameter had a statistically significant association with survival or relapse, suggesting heterogeneity of disease could be an important consideration. 22 % (n=2) of the non-relapsed group and 17% (n=6) of the relapsed group developed acute GvHD. 37% (n= 23) of patients from the whole cohort developed chronic GvHD. Known causes of death included; relapse (36%, n=22), infection (16%, n=10), chronic GvHD (11%, n=7), other (8%, n=5). 26% (n=16) of patients remain alive today and 3% (n=2) were lost to follow-up.Relapse was graded into 5 categories, mirroring monitoring methods at the time of relapse (prior to the development of BCR-ABL PCR). 6% (n=2) of patients developed a molecular relapse (grade 1), 58% (n=21) developed cytogenetic relapse (grade 2), 25% (n=9) developed hematological relapse (grade 3), 3% (n=1) relapsed as accelerated phase CML (grade 4) and 8% (n=3) relapsed with blast phase CML (grade 5). We divided patients into two groups; relapse grade <2 (cytogenetic or molecular relapse only) and grade >3 (hematological relapse or above). In a multivariate Cox proportional model, relapse grades were significantly associated with subsequent survival (log rank p-value 0.0057). Relapse grade >3 had an 11% increased hazard of death compared to relapse grade <2 (Hazard ratio (HR) 1.11; p-value 0.0068). 46% (6/13) of patients with relapse grade >3 underwent a second AHSCT or received DLI, compared with 56% (13/23) of patients with grade <2. In the competing risk analysis, those patients with relapsed grade <2 had a significantly improved survival with a second AHSCT or DLI (HR 1.34; 95% CI 1.06-13.70; p-value 0.03) whereas patients with a relapse grade >3 had no improvement in survival. (HR 0.96; 95% CI 0.93 to 6.8; p-value 0.07, Figure 1).Conclusion:In this cohort, EX-TCD was associated with high rates of relapse. However, patients remain alive from both the non-relapsed and relapsed cohorts. In the relapsed cohort, patients who received a second AHSCT or DLI had improved survival outcomes compared to conservative management. A competing risk analysis demonstrated that patients with lower relapse grades had a statistically significant improvement in survival after a second AHSCT or DLI. DisclosuresMilojkovic:Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Apperley:Novartis: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Efficacy and Safety of Ponatinib in CML and Ph+ ALL Patients in Real-World Clinical Practice: Data from a Belgian Registry

BackgroundPonatinib is a third-generation tyrosine kinase inhibitor (TKI) indicated for adult patients with resistant or intolerant chronic phase (CP), accelerated phase, or blast phase chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), or those with the T315I mutation. In Belgium, ponatinib has been commercially available since March 2016. The goal of this registry was to collect efficacy and safety data in CML and Ph+ ALL patients and to evaluate ponatinib in routine clinical practice in Belgium.MethodsThis ongoing, prospective, multi-center registry includes patients ≥18 years of age with CML or Ph+ ALL, who have initiated ponatinib treatment. Demographic, efficacy and safety data were collected for patients enrolled from March 2016 (day 0) onwards. Results up to study month 24 are presented. Data were analyzed by descriptive statistics. Ethics Committee approval was obtained and all patients provided informed consent.ResultsAt time of data analysis, 34 patients (21 CP-CML and 13 Ph+ ALL) were enrolled. The median age of CP-CML and Ph+ ALL patients was 57 and 55 years, respectively. Patients were heavily pretreated: 90% of CML and 92% of Ph+ ALL patients had received ≥2 prior TKIs. Several patients had one or more risk factors for TKI cardiovascular toxicity: hypertension (10), history of cardiovascular disease (11), smoker (10), hypercholesterolemia (5), and diabetes (4). Median follow-up was 539 days for CML and 135 days for Ph+ ALL patients.The reasons for starting ponatinib therapy were related to refractoriness to previous TKIs (36%), progression (18%), presence of the T315I mutation (18%) or intolerance (29%). Eighty percent (8/10) of the patients who started ponatinib due to intolerance to previous TKIs had received ≥3 prior TKIs. At entry, 17 of the 34 patients (50%) had a confirmed BCR-ABL mutation. Of these 17, 10 (59%; 5 CML and 5 Ph+ ALL) had the T315I mutation. Starting doses of ponatinib in CML patients were 45 mg (76%), 30 mg (10%) and 15 mg (14%) once daily. Starting doses in Ph+ ALL patients were 45 mg (85%), 30 mg (8%) and 15 mg (8%). At latest follow up, the median treatment duration for the 21 CML patients was 531 days (range 15 - 2483) and for the 13 Ph+ ALL patients it was 123 days (range 13 - 1945).Best response was a major molecular response (MMR), which was obtained in 71% of CML patients and 38% of Ph+ ALL patients. The median time-to-best response was 175 days in CML and 35 days in Ph+ ALL patients. In the 10 patients (7 CML and 3 Ph+ ALL) who started ponatinib because of intolerance to several previous TKIs, 80% achieved MMR. The median time to achieve best response in these patients was 192 days for CML and 31 days for Ph+ ALL patients.Treatment-related adverse events (AEs) were reported in 20 patients (59%); the most common were rash (26%), dry skin (9%) and constipation (9%). Three patients reported ≥1 treatment-related serious AE (SAE): thrombocytopenia (n=1), cholecystitis (n=1) and hepatocellular injury (n=1). Three serious cardiovascular events were observed in 1 patient, who had a history of congenital cardiomyopathy and aortic prosthesis. They were scored as not related to ponatinib.Dose reductions or interruptions occurred in 33 cases (20 in CML and 13 in Ph+ ALL patients), with the following reasons most frequently mentioned: AEs (76%), to prevent AEs (18%) and other (6%). Dose increases occurred in 12 cases (10 in CML and 2 in Ph+ ALL patients), for the following reasons: good tolerance of treatment (58%), no or low response (33%) or other (8%). At time of analysis, 19 patients (9 CML and 10 Ph+ ALL) had discontinued treatment, of which 32% due to AEs, 5% due to an SAE, 21% due to planned allogeneic transplant, 16% due to disease progression and 26% due to other reasons.[Note: Percentages may not total 100 due to rounding]ConclusionReal-world evidence from this Belgian registry shows that ponatinib has a favorable efficacy and safety profile in, and supports its use in CML and Ph+ ALL patients who are resistant or intolerant to previous therapies or those with the T315I mutation. Deep molecular responses were obtained in the majority of patients. No new safety signals emerged with ponatinib treatment than those previously reported.Funding: Incyte Biosciences Benelux BV DisclosuresDevos:Takeda: Consultancy; Novartis: Consultancy; Celgene: Consultancy. Theunissen:Incyte: Honoraria. Van Eygen:Janssen: Consultancy, Research Funding; Roche: Research Funding; Amgen: Research Funding. Kuipers:Incyte Biosciences Benelux BV: Employment.

Complete Responses in Relapsed/Refractory Acute Myeloid Leukemia (AML) Patients on a Weekly Dosing Schedule of XmAb14045, a CD123 x CD3 T Cell-Engaging Bispecific Antibody: Initial Results of a Phase 1 Study

Background: Blasts and leukemic stem cells of acute myeloid leukemia (AML) as well as several other hematologic malignancies express CD123, potentially providing a target for novel therapies. XmAb14045 (also known as SQZ622) is a potent bispecific antibody targeting both CD123 and CD3 that stimulates targeted T cell-mediated killing of CD123-expressing cells, regardless of T cell antigen specificity. It is a full-length immunoglobulin molecule designed to be dosed intermittently, in contrast to smaller constructs that are referred to as “DART” or “BiTE” bispecific antibodies that require a continuous infusion.Methods: Patients with relapsed or refractory AML, B cell acute lymphoblastic leukemia (B-ALL), blast phase chronic myelogenous leukemia, or blastic plasmacytoid dendritic cell neoplasm were eligible to enroll on this first-in-human, multicenter, open-label phase 1 dose-escalation study (XmAb14045-01) with standard 3+3 design. The primary objective was to estimate the maximum tolerated dose (MTD) or recommended dose and schedule of XmAb14045. Secondary objectives included safety, preliminary antileukemic activity, and pharmacokinetics/pharmacodynamics of XmAb14045. Treatment was administered weekly in 28 day cycles, using a weight-based dose with a single dose level in Part A and, in Part B, an initial priming dose on Day 1 followed by an escalated dose on subsequent weeks. Premedication to prevent cytokine release syndrome (CRS) was instituted as needed and included a steroid, acetaminophen, and diphenhydramine. Patients were premedicated at all XmAb14045 doses ≥0.075 µg/kg. Therapy was continued for as long as tolerated and there was continuing evidence of therapeutic benefit in the opinion of the investigator. Treatment response was assessed by the 2017 European LeukemiaNet (ELN) criteria after Cycle 1 and after each odd-numbered cycle. CRS was graded using the CRS revised grading system (Lee et al., Blood, 2014).Results: At data cut-off, 64 patients have been treated to date, 63 with relapsed/refractory AML and 1 with B-ALL. Patients had a median age of 61 years and were heavily pretreated (median of 3 prior therapies [range 1-8], including 19 [30%] who had undergone prior allogeneic stem cell transplantation). The recommended dose for Part A was 1.3 µg/kg after a single dose-limiting toxicity of Grade 4 CRS at 2.3 µg/kg; no MTD has been identified. CRS or its component symptoms was the most common treatment-emergent adverse event (TEAE). CRS episodes began within approximately 1-4 hours of the start of drug infusion and occurred in 49 of 64 patients (77%). Seven patients (11%) developed Grade ≥3 CRS, the majority of these on the first dose. There were no CRS-related deaths. Excluding CRS-related events, additional TEAEs occurring in >10% of patients included fatigue (31%), febrile neutropenia (30%), peripheral edema (30%), cough (23%), elevated hepatic transaminases (19%; all recovered without sequelae), pneumonia (17%), stomatitis (14%), hyperglycemia (13%), and sepsis (11%). No myelosuppression requiring dose modification or evidence of tumor lysis syndrome was seen. In Part A, single agent antileukemic activity was documented with a best response of CR (2) or CRi (1) in 3/13 AML patients (CR/CRi rate 23%) treated at the two highest dose levels studied to date (1.3 and 2.3 µg/kg weekly); no CR, CRi, or morphologic leukemia-free state (MLFS) responses were seen at lower doses. Antileukemic activity occurred quickly; all responders had achieved at least an MLFS response after 4 doses (1 cycle). Two responders were bridged to stem cell transplantation, and the third was ineligible for medical reasons but remains in remission at 14+ weeks after initiating therapy.Conclusions: XmAb14045 demonstrated evidence of antileukemic activity in heavily pretreated patients with relapsed/refractory AML treated in Part A at the 1.3 and 2.3 µg/kg doses administered once weekly, with a 23% CR/CRi rate. CRS was the most common toxicity but was generally manageable with premedications. The study is ongoing with further optimization of dose, schedule, and premedication regimen for CRS anticipated during accrual to dose escalation cohorts in Part B.ClinicalTrials.gov Identifier: NCT02730312 DisclosuresRavandi:Xencor: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Sunesis: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Abbvie: Research Funding; Seattle Genetics: Research Funding; Macrogenix: Honoraria, Research Funding; Jazz: Honoraria; Sunesis: Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Orsenix: Honoraria; Xencor: Research Funding; Orsenix: Honoraria; Jazz: Honoraria; Bristol-Myers Squibb: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Macrogenix: Honoraria, Research Funding. Foran:Xencor, Inc.: Research Funding; Agios: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Mawad:Swedish Cancer Institute: Employment. Blum:Astellas: Consultancy; Pfizer: Consultancy; Boehringer Ingelheim: Research Funding; Forma: Research Funding; Xencor: Research Funding; Tolero: Research Funding. Saville:Xencor, Inc.: Employment, Equity Ownership. Johnson:Xencor, Inc.: Employment, Equity Ownership. Vanasse:Novartis: Employment, Equity Ownership. Ly:Xencor, Inc.: Employment, Equity Ownership. Mims:Novartis: Consultancy; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.