AML Subtypes Research Articles

Abstract A56: TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal

Abstract AML with TP53 mutations (TP53Mut) is a poor-risk AML subtype that is largely insensitive to chemotherapy, targeted agents, and hematopoietic stem cell transplantation. TP53Muts are seen in approximately 10-20% of AML and confer a particularly poor prognosis with 1–2-year survival rates to 0-10%. Self-renewal is the defining feature of leukemia stem cells (LSCs) and is central to leukemia progression and relapse. Mutant p53 protein enhances self-renewal in murine models of AML and in human hematopoietic progenitors. In solid tumors, analysis of a variety of TP53Muts defined the proteasome pathway as a common transcriptional target and mediator of oncogenesis among these mutations. Notably, the proteasome degrades I kappa B which activates NF kappa B, which enforces AML self-renewal. Proteasome inhibitors have shown some efficacy in AML clinical trials but have not been well-evaluated in TP53Mut patients. We hypothesize that the proteasome is mediator of self-renewal in TP53Mut AML. To define the transcriptional features of TP53Mut AML, we analyzed the gene expression data in the BEAT AML dataset of primary human AML samples (Tyner, et al. 2018). Relative to TP53WT (n=377 samples), the TP53 mutant samples (n=36) were enriched in every NF kappa B and proteasome gene-set queried. Next, we analyzed a single cell RNA seq dataset of human AML samples (van Galen et al. 2019) and found that the LSC compartment of TP53Mut samples (n=3) strongly upregulated every proteasome gene set we queried (relative to TP53WT samples (n=13)). A recent in vitro screen identified a panel of small molecule inhibitors that reduced viability in TP53-deleted cell lines (Nechiporuk et al. 2019). We tested the activity of these inhibitors in primary human TP53Mut samples. Four of these inhibitors were effective in reducing colony forming capacity or reduced LSC frequency in TP53Mut samples (n=9). We used CyTOF to assess intracellular signaling pathways in LSCs and found that the inhibitors that were most effective in abolishing colony formation and reducing LSC frequency also attenuated NF kappa B levels in TP53Mut LSCs. Accordingly, we found that knockdown of mutant TP53 reduced NF kappa B levels in Kasumi cells (a TP53R248Q AML cell line). Next, we treated a panel of primary human TP53Mut AML samples with the pan-proteasome inhibitors, carfilzomib and bortezomib (FDA approved for lymphoid malignancies) and an immunoproteasome inhibitor, PR957 (in clinical trials for autoimmune conditions). Each drug independently reduced NF kappa B and led to a significant reduction of primary and secondary colony formation. These data suggest that the proteasome/NF kappa B pathway may be an important determinant of self-renewal function in TP53Mut AML and proteasome inhibitors may target the LSCs of this treatment-refractory AML subtype. Citation Format: Marie Lue Antony, Yoonkyu Lee, Klara E Noble-Orcutt, Zohar Sachs. TP53 mutant human AMLs stem cells rely on the proteasome for self-renewal [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A56.

Abstract 6552: ZEB1-AS1 lncRNA prevents ZEB1 suppression and leads to tumor progression in undifferentiated AML subtype

Abstract The zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT). In solid tumors, its overexpression induces EMT, which activates cellular motility and is also associated with the maintenance of stem cell properties, driving tumor progression and metastasis. A double-negative feed-forward loop circuit has been described where, to promote EMT, ZEB1 suppresses the expression of miR-200 family members, which in turn inhibits its activity. Albeit recent evidence highlighted the existence of EMT processes also in hematological malignancies, the role of ZEB1 is not completely defined yet. Hence, this study aims to characterize ZEB1 behavior in acute myeloid leukemia (AML). To this purpose, we retrieved and analyzed RNA-Seq data from public repositories to evaluate the expression levels of the main EMT master regulator genes in normal and malignant hematopoietic cells. We observed a heterogeneous expression of these genes among AML morphological subtypes (from M0 to M5) defined by the French-American-British (FAB) classification system. Specifically, TWIST1 has the highest expression value in M3 and ZEB2 in M5, as already observed in published studies, while ZEB1 has the highest expression in M0, which significantly decreases until M5. To evaluate the ZEB1 transcriptional program, we performed a gene set enrichment analysis of hallmark signatures based on gene-wise correlation with ZEB1 among FABs that revealed the up-regulation of EMT in the M0 subtype. Moreover, the highest value of correlation was found between ZEB1 and its antisense lncRNA (ZEB1-AS1). This trait emerged as peculiar to AML both with respect to other cancer types and normal hematopoietic cells. Since ZEB1 orchestrates its transcriptional program by inhibiting the activity of specific miRNAs, we selected putative candidates that may take part in this circuit in AML by calculating their bimodality distribution, which reflects the toggle switch behavior typical of the double-negative feed-forward loop. We identified miR-425-5p, which was coherently down-regulated in M0 with respect to other FABs, and miR-151-3p which was surprisingly up-regulated. Specifically, the simultaneous up-regulation in the M0 subtype of miR-151-3p and its target ZEB1 is inconsistent with the toggle switch behavior of the circuit. Thus, we hypothesized that miR-151-3p is not able to inhibit ZEB1 expression in M0 due to the presence of ZEB1-AS1, which is supposed to disrupt the circuit mechanism protecting ZEB1 from miRNA-mediated inhibition. This hypothesis is also supported by the fact that all target genes of miR-151-3p were significantly up-regulated. A better comprehension of this regulatory circuit could allow identifying aberrant epigenetic mechanisms during leukemic development and discriminating of the different AML subtypes. Citation Format: Stefano Percio, Daniela Magliulo, Silvia Martini, Sandro Pasquali, Rosa Bernardi. ZEB1-AS1 lncRNA prevents ZEB1 suppression and leads to tumor progression in undifferentiated AML subtype. [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 6552.

Explainable AI identifies diagnostic cells of genetic AML subtypes.

Explainable AI is deemed essential for clinical applications as it allows rationalizing model predictions, helping to build trust between clinicians and automated decision support tools. We developed an inherently explainable AI model for the classification of acute myeloid leukemia subtypes from blood smears and found that high-attention cells identified by the model coincide with those labeled as diagnostically relevant by human experts. Based on over 80,000 single white blood cell images from digitized blood smears of 129 patients diagnosed with one of four WHO-defined genetic AML subtypes and 60 healthy controls, we trained SCEMILA, a single-cell based explainable multiple instance learning algorithm. SCEMILA could perfectly discriminate between AML patients and healthy controls and detected the APL subtype with an F1 score of 0.86±0.05 (mean±s.d., 5-fold cross-validation). Analyzing a novel multi-attention module, we confirmed that our algorithm focused with high concordance on the same AML-specific cells as human experts do. Applied to classify single cells, it is able to highlight subtype specific cells and deconvolve the composition of a patient's blood smear without the need of single-cell annotation of the training data. Our large AML genetic subtype dataset is publicly available, and an interactive online tool facilitates the exploration of data and predictions. SCEMILA enables a comparison of algorithmic and expert decision criteria and can present a detailed analysis of individual patient data, paving the way to deploy AI in the routine diagnostics for identifying hematopoietic neoplasms.

Targeting HDAC3 to overcome the resistance to ATRA or arsenic in acute promyelocytic leukemia through ubiquitination and degradation of PML-RARα.

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML-RARα, which recruits corepressor complexes, including histone deacetylases (HDACs), to suppress cell differentiation and promote APL initiation. All-trans retinoic acid (ATRA) combined with arsenic trioxide (ATO) or chemotherapy highly improves the prognosis of APL patients. However, refractoriness to ATRA and ATO may occur, which leads to relapsed disease in a group of patients. Here, we report that HDAC3 was highly expressed in the APL subtype of AML, and the protein level of HDAC3 was positively associated with PML-RARα. Mechanistically, we found that HDAC3 deacetylated PML-RARα at lysine 394, which reduced PIAS1-mediated PML-RARα SUMOylation and subsequent RNF4-induced ubiquitylation. HDAC3 inhibition promoted PML-RARα ubiquitylation and degradation and reduced the expression of PML-RARα in both wild-type and ATRA- or ATO-resistant APL cells. Furthermore, genetic or pharmacological inhibition of HDAC3 induced differentiation, apoptosis, and decreased cellular self-renewal of APL cells, including primary leukemia cells from patients with resistant APL. Using both cell line- and patient-derived xenograft models, we demonstrated that treatment with an HDAC3 inhibitor or combination of ATRA/ATO reduced APL progression. In conclusion, our study identifies the role of HDAC3 as a positive regulator of the PML-RARα oncoprotein by deacetylating PML-RARα and suggests that targeting HDAC3 could be a promising strategy to treat relapsed/refractory APL.

Cre recombinase expression cooperates with homozygous FLT3 internal tandem duplication knockin mouse model to induce acute myeloid leukemia

Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration. We report on a transgenic murine model of FLT3-ITD induced disease, where Cre recombinase expression alone, and in the absence of a conditional allele, gives rise to an aggressive leukemia phenotype. Here, expression of various Cre recombinases leads to polyclonal expansion of FLT3ITD/ITD progenitor cells, induction of a differentiation block and activation of Myc-dependent gene expression programs. Our report is intended to alert the scientific community of potential risks associated with using this specific mouse model and of unexpected effects of Cre expression when investigating cooperative oncogenic mutations in murine models of cancer.

Prospective Evaluation of Sorafenib Combined with Chemotherapy in Newly Diagnosed Adult Core-Binding Factor Acute Myeloid Leukemia: An Open-Label, Randomized Controlled, Multicenter Phase II Trial

Objective Core-binding factor acute myeloid leukemia (CBF-AML) includes AML with t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22) chromosomal rearrangements, leading to the RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes, respectively. Although CBF-AML is considered to have a favorable prognosis relative to other AML subtypes, up to 40% of these patients experience relapse. Recent studies demonstrate achievement of complete molecular remission (CMR) at the end of treatment significantly associated with a reduced risk of relapse. A high frequency of mutations and/or high expression of KIT gene is observed in CBF-AML. Sorafenib is a first-generation type-II multitargeted tyrosine kinase receptor inhibitor (TKI) that suppresses various signaling pathways associated with the development of AML, such as RTK (FLT3, c-KIT). Thus the purpose of this study is to evaluate the safety and efficacy of sorafenib combined with chemotherapy in adult newly diagnosed CBF-AML. Methods Patients were randomly assigned (1:1) to control group (chemotherapy alone) or sorafenib group (sorafenib combined with chemotherapy). For control group, patients received one cycle of induction therapy with idarubicin (12 mg/m² on days 1-3) plus cytarabine (100 mg/m² on days 1-7), followed by one cycle of idarubicin (8 mg/m² on days 1-3) plus cytarabine (2 g/m² q12h on days 1-3) and two cycles of high-dose cytarabine consolidation therapy (2 g/m² q12h on days 1-3). For sorafenib group, patients were treated with chemotherapy plus sorafenib at 400 mg twice daily on days 8-21 for induction cycle, and on days 1-21 for each consolidation, and as maintenance for 12 months. Allogeneic or autologous HSCT were implemented based on the measurable residual disease (MRD) level and donor availability after 4 cycles of therapy. MRD levels were serially monitored for RUNX1-RUNX1T1 or CBFB-MYH11 transcripts by real-time quantitative polymerase chain reaction. Results were expressed as a [fusion gene/ABL1] ×100 transcript ratio. Major molecular response (MMR) and complete molecular remission (CMR) are defined as transcript ratio < 0.1% and 0.001%, respectively. The primary endpoint was CMR after 4 cycles of therapy in bone marrow (BM). Results Between January 2020 and March 2022, 64 patients were enrolled and randomly assigned to sorafenib group (n=32) or control group (n=32). There is no significant difference between the two groups about age, gender, CBF subtype, BM blast percentage, additional chromosome abnormalities and gene mutations at diagnosis. The hematological complete remission in the sorafenib and control group were 96.9% and 94.1%, respectively (p=.592). Following three cycles of therapy, MMR was observed in 79.3% of the sorafenib group and 46.9% of the control group (p =.009). Sorafenib group also had a significantly higher CMR than control group (62.1% vs 28.1%, p =.009). After four cycles of therapy, MMR increased to 100% in the sorafenib group and 70.8% in the control group (p =.019), whileCMR was also more frequently achieved in the sorafenib group than that in the control group (90.9% vs 54.2%, p =.006). There was no significant difference of grades 3-4 adverse events between the two groups. Grade 4 neutropenia (neutrophil count less than 0.5×10⁹ cells per L) and thrombocytopenia (platelet count less than 20×10⁹ per L) were observed in all cases. The median duration of neutropenia was 12d (range, 6-25d) in the sorafenib group and 10d (range, 5-22d) in the control group (p =.063) , while the median duration of thrombocytopenia was 12d (range, 5-33d) and 9d (range, 6-19d) (p =.052), respectively.The most common grade 3-4 non-hematological adverse events in sorafenib and control group were pneumonia (31.3% vs 57.4%, p=.019), mucositis (21.9% vs 24.1%, p =.816), blood stream infection (37.5% vs 31.5%, p =.568). Conclusion Sorafenib combined with chemotherapy significantly increased the CMR after 4 cycles of therapy, however, the incidence of haematological and non-haematological adverse events did not apparently increased. The impact of sorafenib combined with chemotherapy on relapse and survival of CBF-AML need further following-up. Keywords: Sorafenib, CBF-AML, CMR, adverse events

Access and Treatment Equity across the Comprehensive Cancer Center Catchment Area: Targets for Disparities Remediation in Acute Leukemia Research Participation

Introduction Race-ethnic disparities in acute leukemia research participation limit generalizability and equitable access to novel therapies. We recently found substantial clinical trial enrollment disparities between National Cancer Institute-designated Comprehensive Cancer Center (CCC) hospitals and the catchment areas for which they assume cancer control and treatment responsibility (Hantel JCO 2022). A first step in remediating such participatory disparities is understanding how underserved populations access care at these hospitals. Methods We analyzed hospital access and initial treatment data for a large CCC hospital, the Dana-Farber Cancer Institute (DFCI), relative to its catchment area, Massachusetts (MA), for all patients diagnosed with acute myeloid or lymphoblastic leukemia between 2014-2018. DFCI data were from an institutional cancer registry (OncDRS) that includes rich demographic, treatment, and outcome data; state data were from the MA Cancer Registry. We assessed access proportions (2+ visits for leukemia care) between mutually exclusive race-ethnic groups, using X2 testing, as patients moved from the incident populations within and outside the catchment area to the hospital, and based on their place of initial treatment. Non-Hispanic Whites (NHW) were the comparator with odds ratios (OR) and 95% confidence intervals (CI) reported. For some comparisons, race-ethnicity of was dichotomized as NHW and Diverse (Hispanic White and Hispanic- or non-Hispanic-Black, -Asian/Pacific Islander, -Native American, -another race, and -two or more races). We performed logistic regression modeling of demographics, social determinants of health, and disease characteristics (Table 1) to assess their association with DFCI as the site of initial treatment using complete cases. Results From 2014-18 there were 2077 incident cases of acute leukemia in MA, of which 858 (41.3%) were seen at DFCI. During the same period, 1622 patients with acute leukemia accessed care at DFCI, of which 764 (47.1%) were from outside MA; DFCI patient characteristics are shown in Table 1. Hospital access and location of initial treatment proportions by leukemia subtype, race-ethnicity, and place of residence are shown in Figure 1: bar charts show differences for race-ethnic groups relative to MA incidence proportions, which are displayed in the pie charts. Relative to MA incidence, there was no significant difference in DFCI access by dichotomized race-ethnicity (Diverse vs NWH OR 0.87; 95% CI 0.73,1.05). Among MA residents, NHWs were not more likely to access DFCI than those from Diverse race-ethnicities (OR 0.82; 95% CI 0.65,1.04). NHW were more likely to have received initial treatment at DFCI (OR 1.59; 95% CI 1.12,2.28). Conversely, patients accessing DFCI from outside MA were more likely to be NHW (OR 1.94; 95% CI 1.42,2.65) but were equally likely to have received initial treatment at DFCI (OR 1.04; 95% CI 0.62,1.74). In a multivariable model assessing site of initial treatment receipt for those having accessed DFCI, including only variables from Table 1 that univariably predicted initial treatment site (with predicted defined as p<0.05: age, race-ethnicity, leukemia subtype, alcohol and tobacco use, marital status, primary language, and primary insurer), NHW race-ethnicity was not independently associated with initial treatment receipt (OR 0.69; 95%CI 0.47,1.02) but older age (OR 1.02; 95%CI 1.01,1.03), AML subtype (OR 2.62; 95%CI 1.91,3.95), and primary Medicare insurance (OR 1.70 vs private; 95%CI 1.24,2.31) were. Conclusions For this large and high-enrolling CCC hospital, overall access to leukemia care did not significantly differ by race-ethnicity. This welcome finding suggests that participatory equity for acute leukemia clinical research is achievable at such sites. Known participatory disparities, however, are significant and prevalent. The disparities in access from outside the catchment area and initial treatment within it define targetable issues that impact the ability of under-enrolled populations to participate in clinical research equitably. Next steps include correlating access and enrollment data and developing real-time participatory diversity monitoring tools. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myeloid Neoplasms with MYC-Positive Double Minutes, a Specific Subgroup?

Background: Double minutes (dmin) are a rare cytogenetic finding in hematologic malignancies, mostly associated with acute myeloid leukemias. The acentric chromosomal structures usually represent the cytogenetic equivalent of the amplification of an oncogene, such as MYC. Due to their small size dmin are difficult to detect in chromosome banding analysis (CBA) and FISH analysis is required to identify the corresponding gene amplification. In literature several case reports described an association of the presence of MYC-positive dmin with an APL-like cytomorphology, however, larger studies comprehensively characterizing these cases are missing so far. Aim: Characterization of 68 cases of myeloid neoplasms harboring MYC dmin with respect to cytogenetics, morphology, gene expression (GE) and gene mutations. Patients and methods: The study enrolled 68 patients (pts) with myeloid neoplasia and MYC dmin. The diagnosis was established following WHO guidelines. Dmin and MYC amplification were assessed via CBA and FISH. Cases were screened for morphological features indicating a similarity to APL and/or suspected diagnosis of APL (APL-like). NGS of a 72 gene panel and WTS (50 Mio reads) were performed on NovaSeq instruments. Variant calling was performed with Pisces (minimum sensitivity: 3 %). Results: The majority of pts were diagnosed with AML (52/68, 77 %, 7 of these secondary AML) or MDS/AML (3/68, 4 %). Other diagnoses comprised CMML (4/68, 6 %), MPN in acceleration or blast crisis, MDS/MPN overlap or MDS (3/68, 4 % each). APL-like cases amounted to 17/68 (25 %). Auer rods were detected in 24/68 pts (35 %) with a higher frequency in APL-like cases (p=0.007). Myeloperoxidase level was >75 % in 46/54 pts (85 %). CBA revealed a complex karyotype (ck, ≥3 aberrations in addition to MYC dmin) in 21/68 pts (31 %), whereas 19/68 pts (28 %) presented with MYC dmin as the only cytogenetic aberration (MYC dmin only). Most common additional chromosomal gains were +4 (11/68, 16 %), +6 (9/68, 13 %) and +22 (8/68, 12 %), while most frequent chromosomal losses comprised 5q deletions (19/68, 28 %), 17p deletions (18/68, 27 %) and 9q deletions or -9 (16/68, 24 %). Gene mutations (mut) most frequently occurred in TET2 (48/68, 71 %), TP53 (22/67, 33 %), U2AF1 (17/67, 25 %) and NRAS (12/66, 18 %). Almost all pts (66/68, 97 %) harbored at least one mutation in TET2, TP53 or U2AF1. TP53 mut were associated with ck (p<0.001) and were not detected in MYC dmin only pts (p<0.001). EZH2 mut were exclusively observed in cases with non-ck and 5/7 mut occurred in MYC dmin only pts (p=0.014). TET2 mut were observed more frequently in cases with non-ck (89 % vs. 29 %, p<0.001). Similarly, U2AF1 mut co-occurred with ck only in one case (p=0.013). In addition, U2AF1 showed significantly higher mutation frequency in APL-like cases (47 % vs. 18 %, p=0.025). A comparison of the mutation frequencies of all MYC dmin cases to a cohort of AML from routine diagnostics (707 pts) revealed overrepresentation of mut in TP53 (33 % vs. 11 %, p=0.003), TET2 (71 % vs. 19 %, p<0.001), U2AF1 (25 % vs. 4 %, p<0.001) and EZH2 (11 % vs. 2 %, p<0.001) in the cohort of MYC dmin cases, whereas CEBPA, NPM1, FLT3, FLT3-ITD and WT1 mut were only detected in the AML cohort (10 %, p=0.004; 22 %, p<0.001; 8 %, p=0.011, 16 %, p<0.001 %; 8 %, p=0.017 respectively) and IDH2 mut occurred less frequently in MYC dmin cases (2 % vs. 9 %, p=0.022). GE analysis of 40 MYC dmin pts revealed high expression of MPO compared to various AML subtypes, similar to APL (Figure (A)). A tSNE plot based on the top 5 % most variable genes demonstrated similar GE profiles within the MYC dmin cases compared to other AML cases (857 pts) (Figure (B)). A comparison of GE profiles within the MYC dmin cohort revealed differential GE between APL-like and all other MYC dmin cases (46 differentially expressed genes (DEG), adjusted p-value <0.05), as well as between ck and non-ck MYC dmin cases (106 DEG, adjusted p-value <0.05). Conclusions:MYC dmin cases often show an APL-like cytomorphology, Auer rods and high levels of MPO expression. They are characterized by a high frequency of TET2, TP53 and U2AF1 mut as well as a relatively uniform GE profile. Mutation patterns and GE profiles allow further differentiation within MYC dmin cases. In cases with suspected APL after exclusion of a PML::RARA or other RARA fusions CBA and FISH for the detection of MYC dmin is helpful to identify this rare myeloid subtype which is described to be associated with an unfavorable prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of Newly Diagnosed Therapy-Related AML and AML with Myelodysplasia-Related Changes Treated with 7+3, Hypomethylating Agents with or without Venetoclax and CPX-351: A Retrospective Cohort Study

Introduction Therapy-related acute myeloid leukemia (t-AML) and AML with myelodysplasia-related changes (AML-MRC) are associated with poor outcomes due to older age at diagnosis and adverse-risk cytogenetics and molecular features and, therefore, treatment of these groups of patients remain a challenge. There is a paucity of real-world data on outcomes of t-AML and AML-MRC treated with 7+3, hypomethylating agents (HMA) with or without venetoclax (VEN) or CPX-351 (Vyxeos). Methods We conducted a single-center retrospective cohort study to compare overall survival (OS), event-free survival (EFS), and composite complete response (CCR) of patients with t-AML or AML-MRC treated with first-line 7+3, HMA, HMA+VEN or Vyxeos. Therapy-related myelodysplastic syndrome were not included. OS was defined from day 1 of initial chemotherapy until death from any cause. Patients were not censored at the time of allogeneic transplant. Sixty and 90-day treatment-related mortality were estimated as surrogates for treatment toxicity. Propensity score modeling was used to adjust for baseline characteristics (age, AML subtype, comorbidities, performance status, cardiac ejection fraction, cytogenetics, and mutations). Results Of 488 consecutive AML patients treated at the University of Maryland between 2013-2021, 136 patients had t-AML or AML-MRC and received one of the above therapies. 40% were female. The median age of the cohort was 67.5 [IQR: 60.9-73.2]. The median age of patients who received 7+3 (n=34) was 63.4 years, HMA (n=61) 70.3 years, HMA+VEN (n=27) 72.6 years, and Vyxeos (n=14) 62.2 years (age P-value= 0.1). 42 and 17 patients received decitabine in the HMA and HMA+VEN arms, respectively. None of the patients treated with 7+3 or Vyxeos received HMA prior to AML diagnosis. However, 3 and 6 patients received HMA in the HMA and HMA+VEN arms prior to AML diagnosis, respectively. Both 7+3 and HMA were given mostly before 2019 (7+3: 88%; HMA: 81%) compared to HMA+VEN and Vyxeos, which were given mostly in years 2019 and after (HMA+VEN: 74%; Vyxeos: 71%) (P<0.01). The median follow-up was 73.5 months (mo). Table 1 demonstrates unadjusted outcomes. Figure 1 demonstrates the unadjusted OS of treated groups. Using propensity score for adjustment, there was no difference in median OS or CCR in patients treated with 7+3 vs. HMA (11.2 vs. 10.6 mo, P=0.63; 42% vs. 31%, P=0.38). However, patients treated with HMA+VEN had worse adjusted m-OS than patients treated with 7+3 (5.3 vs. 20.2 mo, P=0.02) or HMA alone (5.3 vs. 10.1 mo, P=0.02). Patients treated with Vyxeos had a statistically similar adjusted-median OS to those treated with 7+3 (5.1 vs. 15.4 mo, P=0.34) and a trend toward a worse outcome than those treated with HMA alone (5.3 vs. 10.1 mo, P=0.07). Conclusion The outcomes of t-AML and AML-MRC remains poor. In t-AML and AML-MRC, using 7+3 and HMA resulted in similar OS; however, improved outcomes compared to HMA+VEN. Almost 50% of patients treated with HMA+VEN died in the first 90 days, possibly related to treatment toxicity. Further studies are needed to determine the role of VEN combination with HMA in t-AML and AML-MRC. Moreover, further real-world, multi-institutional data are needed to evaluate the effectiveness of Vyxeos in t-AML and AML-MRC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Anti-Leukemic Activity of STRO-002 a Novel Folate Receptor-α (FR-α)-Targeting ADC in Relapsed/Refractory CBF2AT3-GLIS2 AML

BACKGROUND: CBF2AT3-GLIS2 (CBF/GLIS) oncogenic fusion is the underlying genomic cause of "RAM" phenotype AML, a megakaryoblastic subtype exclusively seen in infants and young children. CBF/GLIS AML is a highly refractory and uniformly fatal subtype of AML with limited response to conventional chemotherapy. We have demonstrated that FR-α, a known target in ovarian cancer, is expressed in CBF/GLIS AML cells and this expression is causally linked to the fusion oncogene. STRO-002 is currently under clinical investigation for the treatment of relapsed/refractory ovarian and endometrial cancer (NCT03748186). Preclinical studies have tested and confirmed the potent in vitro and in vivo anti-leukemia activity of STRO-002 in CBF/GLIS AML cell lines and xenograft models (Le et al. ASH 2021). Based on these compelling preclinical pharmacology observations, Sutro Biopharma evaluated patients on a case-by-case basis for consideration of a single patient IND (compassionate use) program for pediatric patients with CBF/GLIS AML. METHODS: Key criteria for consideration of STRO-002 were CBF2AT3-GLIS2 oncogenic fusion and FR-α expression by flow cytometry in AML sample. Key exclusion criteria were co-existent acute/uncontrolled infection and compromised organ function (pulmonary, hepatic, renal, etc). Between August 2021 and July 2022, 16 patients received STRO-002. Median age at treatment was 2 years (range 6 months-7 years). Median number of prior therapies was 3 (range 1-8). Eight patients had relapsed after stem cell transplant (SCT) and the remaining 8 had primary refractory (induction failure) or relapsed AML. RESULTS: In the 16 patients treated, 10 received STRO-002 as monotherapy, and 6 received combination with chemotherapy (fludarabine/cytarabine, decitabine, methotrexate (MTX), or dasatinib). All 16 pts were evaluable for response, best observed response included 7 CRs with 6 MRD negative remissions. Of these CRs, 3 occurred after monotherapy and 4 with combination therapy: MTX/fludarabine/cytarabine (n = 1), decitabine/DLI (n = 1), fludarabine/cytarabine/G-CSF (n = 1) and dasatinib/DLI (n = 1). The remaining 9 patients had stable disease or transient decrease in disease burden. Of the 7 patients who achieved a CR, 4 proceeded to SCT, 2 are receiving DLI and 1 will receive consolidation therapy. Ten (63%) of the 16 evaluable patients are alive and the other 6 died of progressive disease. Follow-up is limited for the surviving patients. Of note were 6 patients who achieved an MRD negative CR, two with primary refractory disease, and four who had relapsed after an allogeneic SCT. These six patients with significant disease had deep response to STRO-002 with MRD negative remission. One patient had a rapid decline in marrow blasts and had no evidence of disease by flow after 3 single agent every other week (QOW) doses of STRO-002. A second patient had a similarly rapid response to single agent STRO-002 with clearance of all detectable disease after a second QOW dose of STRO-002. ANC for both patients one week after last dose of STRO-002 was >1000. These patients were consolidated with allogeneic stem cell transplant or with DLI. STRO-002 was generally well tolerated. Collection and review of treatment-emergent AEs (TEAEs) is ongoing. Treatment-related events ≥ grade 3 included neutrophil count decreased (n = 3), platelet count decreased (n = 3), anemia (n = 2), WBC decreased (n = 2), hyperbilirubinemia (n = 1), bacteremia (n = 1), febrile neutropenia (n = 1), urticaria (n = 1), generalized edema (n = 1), aspartate aminotransferase elevation (n = 1), alanine aminotransferase elevation (n = 1). CONCLUSIONS: STRO-002 has promising activity in relapsed/refractory CBF2AT3-GLIS2 AML, a disease that tends to be highly refractory to all standard-of-care (SOC) therapies. Further, STRO-002 is well tolerated as a monotherapy agent and in combination with SOC therapies. Patients with low tumor burden were more likely to achieve CR and transition to SCT, DLI and CAR-T. Deep molecular and flow cytometric remission was achieved in a cohort of patients. Patients with primary refractory disease (no prior transplant), 1-3 prior lines of therapy, low disease burden, or without extramedullary disease appeared to have a more potent response to STRO-002. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371

AbstractAcute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications–driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

New Mutation Identified May Define High-Risk Subtype of Pediatric AML

New Mutation Identified May Define High-Risk Subtype of Pediatric AML

The Development and Clinical Applications of Oral Arsenic Trioxide for Acute Promyelocytic Leukaemia and Other Diseases.

Appreciation of the properties of arsenic trioxide (ATO) has redefined the treatment landscape for acute promyelocytic leukaemia (APL) and offers promise as a treatment for numerous other diseases. The benefits of ATO in patients with APL is related to its ability to counteract the effects of PML::RARA, an oncoprotein that is invariably detected in the blood or bone marrow of affected individuals. The PML::RARA oncoprotein is degraded specifically by binding to ATO. Thus ATO, in combination with all-trans retinoic acid, has become the curative treatment for ATO. The multiple mechanisms of action of ATO has also paved the way for application in various condition encompassing autoimmune or inflammatory disorders, solid organ tumours, lymphomas and other subtypes of AML. The development of oral formulation of ATO (oral ATO) has reduced costs of treatment and improved treatment convenience allowing widespread applicability. In this review, we discuss the mechanisms of action of ATO, the development of oral ATO, and the applications of oral ATO in APL and other diseases.

Dissecting super-enhancer heterogeneity: time to re-examine cancer subtypes?

The heterogeneity of transcriptional regulations by super-enhancers (SEs) is poorly understood in human cancers. Herein, we summarize a bioinformatics workflow for genome-wide SE profiling and identification of subtype-specific SEs and regulatory networks. Dissecting SE heterogeneity provides new insights into cancer biology and alternative therapeutic strategies for cancer precision medicine.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are...

P508: REAL LIFE EXPERIENCE USING FRONT-LINE CPX-351 FOR THERAPY-RELATED AND AML-MRC: RESULTS FROM THE SPANISH PETHEMA REGISTRY.

Background: Acute Myeloid Leukemias arising after cytotoxic therapy (t-AML) or with myelodysplasia-related changes (AML-MRC) share adverse risk features and poor outcomes after standard 3 + 7 chemotherapy. In a randomized clinical trial, CPX-351 has shown superior overall survival (OS) compared to standard anthracycline-cytarabine schedule in these AML subtypes. Aims: to evaluate the effectiveness of CPX-351 treatment in a real-world setting. Methods: adult t-AML and AML-MRC patients who have been treated upfront with CPX-351 in 35 Spanish centers between 2018 and 2021. All patients were included in the PETHEMA registry (NCT02606825). Primary end-point was OS. Secondary end points were complete remission with or without hematological recovery (CR/CRi), proportion of minimal residual negativity (MRD), rate of allogeneic hematopoietic stem cell transplant (HSCT) and safety. Results: CPX-351 was administered to 74 patients as first induction chemotherapy. Median age was 67 (63-71) years, with 40% (30/74) of female patients. ECOG performance status score was 0-1 in 85% (53/62) patients. A diagnosis of t-AML was present in 30% (22/74) patients. Hypomethylating agents were used for MDS or CMML phase in 15%. Hematopoietic Transplant Comorbidity Index was low, intermediate and high in 14% (10/74), 43% (32/74) and 43% (32/74) patients. ELN17 risk classification was favourable, intermediate, adverse and indeterminate in 9% (7/74), 38% (28/74), 46% (34/74) and 5% (4/74). CR/CRi was obtained in 43% (32/74) patients after first cycle with CPX-351. Three additional patients achieved Morphological Leukemia Free Status. A second induction with CPX-351 was administered in 5 patients with 100% CR (CR/CRi after 2 cycles 37/74, 50%). MRD was evaluated in 97% (36/37) of CR/CRi patients, being low (<0.1%) in 47% (17/36) of them. In univariate analysis, no factor was associated with response. However, 71% (5/7) response rate was observed in the favourable ELN17 genetic risk group compared to 48% (32/67) in the non-favourable. HSCT was performed in 27% (20/74) patients, in 1st CR/CRi in 70% (14/20), MLFS in 15% (3/20) and active disease in 10% (2/20). With median follow up of 11.9 months (7.2-23.6) from HSCT, median OS was not reached (95% CI 9.5-NR months (Figure). In univariate analysis no factors were significantly associated with OS after HSCT. Median time to absolute neutrophil count ≥0.5 x109/L and platelets ≥50 x109/L were 30.5 (25-35) and 30 (26-39) days, respectively. Deaths within the first 30 days after induction occurred in 12% (9/74) patients. Causes of early death: infection in 5 patients, haemorrhage in 3 and respiratory failure in 1. With 6.1 (1.8-14) months of median follow up, median OS was 10.5 (6.9-NR) months, with significant differences between HSCT and non-HSCT patients [NR, (95% CI: 12.7-NR), vs. 6 months, (95% CI: 4-NR), P<0.001]. In univariate analysis, factors associated with lower OS were non-favourable ELN17 [8.9 vs 23.5, P=0.04]; ECOG≥2 [0.7 vs 12., P=0.002]; age ≥65 [7.9 vs NR, P=0.007] and male [6.6 vs NR, P= 0.007]. In multivariate analysis, age above 65 years [HR 1.6, P<0.001], male [HR -0.9, P=0.02] and ECOG≥2 [HR 1.9, P<0.001] were associated with OS. Image:Summary/Conclusion: Our real-life cohort was comparable to the target population of the pivotal trial including patients diagnosed with sAML (t-AML, AML-MRC and ELN17 high risk AML), obtaining similar outcomes than the CPX-351 phase 3 trial arm. To optimize treatment outcomes CPX-351 should be preferably offered to t-AML and AML-MRC patients who are suitable for HSCT.

P463: RE-PURPOSING OF GENE SIGNATURES IN AML UNCOVERS NOVEL ENERGETICS-ASSOCIATED MOLECULAR SUBTYPES

Background: AML is a highly heterogeneous disease with great diversity in clinical features and patient response to treatments. Despite recent improvements in disease understanding, treatments have remained unchanged for 30 years. This presents the need to characterise the disease more fully and characterise its underpinning molecular subtypes. The re-use of published and validated prognostic and predictive gene signatures e.g. Cancer Hallmarks presents an invaluable in silico opportunity to uncover the biological mechanisms underpinning treatment response in AML. Aims: To develop an automated statistical analysis pipeline combined with machine learning techniques to derive robust patient clusters representative of novel molecular AML subtypes significantly associated with clinical variables and survival outcomes. Methods: Analysis was carried out using a primary dataset (AML-OHSU: 451 AML patient samples) and a validation dataset (TCGA-LAML: 200 AML patient samples). Both datasets had been processed using Almac’s claraT platform, a software-driven solution which provides a comprehensive overview of tumour profiles using gene expression signatures. An automated analytical pipeline was developed using Consensus Clustering (CC), a method that determines the number and membership of potential clusters, using different combinations of 8 distances and 7 linkages within a dataset. Robustness was tested via bootstrapping. This pipeline was used to stratify patients via a dimension reduction approach whereby clustering was performed on 210 gene signatures categorised by 10 different hallmarks of cancer. Analysis was performed to identify clinical associations within robust clusters that were linked to differences in survival. Results: The automated clustering pipeline analysed a total of 1,314 stable clusters across 10 cancer hallmarks in the AML-OHSU dataset. Stable clusters were subsequently processed via log rank analysis (OS right-censored at 60 months) identifying 134 stable clusters with significant differences (p-value <0.05) in survival outcome. Stable clusters with significant survival differences were tested against 32 clinical categorical variables present in the AML-OHSU dataset. The results were filtered for a significant threshold (chi square p-value <0.05 and BH p-value <0.2). Here we found gene signatures representative of the Energetics hallmark, incorporating 22 signatures, to be one of the most frequently clustered throughout our results, ranking highest where K=3. A significant difference in overall survival probability (Log rank p-value: 0.033) was found between clusters (Energetics, K=3). Patients in the poorest survival cluster were characterised by a refectory induction response to treatment, having the lowest number of fusions, a low frequency of NPM1 mutations and a high proportion of patients above the age of 65. To validate energetics results from the AML-OHSU dataset, CC was again performed using gene signatures from the TCGA dataset that were representative of the energetics hallmark. A significant difference between overall survival probability (Log rank p-value: 0.019) was again found between stable clusters of the energetics hallmark (K = 3). Summary/Conclusion: We have demonstrated that a novel analytical pipeline developed here to analyse Hallmark-related gene signatures can aid in the discovery of new molecular subtypes in AML associated with prognosis. We have subsequently validated these in an independent dataset. The Energetics hallmark has not, to our knowledge, been linked with AML prognosis before, and may suggest novel biology linked to treatment response.

ATRA use in Acute Promyelocytic Leukemia

Acute Leukemia is a the result of malignant event or events occurring in early hematopoietic precursor, instead of proliferation and differentiation normally, the affect cells give rise to progency that fail to differentiate and instead continue to proliferate in an uncontrolled fashion. Retrospective study, carried between January 2001, and August 2006, Ninety four children ( 15 years of ages) were diagnosed as AML by bone marrow examination (aspirate and sometimes biopsy), twenty two of these children diagnosed as APL, fourteen were treated by chemotherapy and eight of these children treated by ATRA and specific protocol. M 2 subtype of AML is most common subtype. M 3 second most common subtype of AML with peak age of frequency 10-11 years with female predominance. Combined ATRA with chemotherapy for treatment of APL improves survival rate of children. The most common complication of ATRA encounter during treatment was dryness of skin and mucosa.

Acute Leukemia Classification Using Transcriptional Profiles From Low-Cost Nanopore mRNA Sequencing.

Most cases of pediatric acute leukemia occur in low- and middle-income countries, where health centers lack the tools required for accurate diagnosis and disease classification. Recent research shows the robustness of using unbiased short-read RNA sequencing to classify genomic subtypes of acute leukemia. Compared with short-read sequencing, nanopore sequencing has low capital and consumable costs, making it suitable for use in locations with limited health infrastructure. We show the feasibility of nanopore mRNA sequencing on 134 cryopreserved acute leukemia specimens (26 acute myeloid leukemia [AML], 73 B-lineage acute lymphoblastic leukemia [B-ALL], 34 T-lineage acute lymphoblastic leukemia, and one acute undifferentiated leukemia). Using multiple library preparation approaches, we generated long-read transcripts for each sample. We developed a novel composite classification approach to predict acute leukemia lineage and major B-ALL and AML molecular subtypes directly from gene expression profiles. We demonstrate accurate classification of acute leukemia samples into AML, B-ALL, or T-lineage acute lymphoblastic leukemia (96.2% of cases are classifiable with a probability of > 0.8, with 100% accuracy) and further classification into clinically actionable genomic subtypes using shallow RNA nanopore sequencing, with 96.2% accuracy for major AML subtypes and 94.1% accuracy for major B-lineage acute lymphoblastic leukemia subtypes. Transcriptional profiling of acute leukemia samples using nanopore technology for diagnostic classification is feasible and accurate, which has the potential to improve the accuracy of cancer diagnosis in low-resource settings.

Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness

AbstractAcute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.