Philadelphia Chromosome-like Acute Lymphoblastic Leukemia Research Articles

Abstract 6618: Pleiotropic GATA3 locus is associated with multiple childhood cancers: harnessing the existing data from the St. Jude Lifetime Cohort and the Childhood Cancer Survivor Study for genetic etiology research

Abstract Background: A genetic variant (rs3824662) at GATA3 was previously associated with risk of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) and relapse. Our recently published childhood Hodgkin lymphoma (HL) study identified a genome-wide significant association for another genetic variant (rs3781093) in high linkage disequilibrium (D’=1.0, R2=0.91) with rs3824662. We hypothesize that GATA3 is a pleiotropic locus associated with multiple childhood cancers. Methods: The study was performed by combining 3 cohorts of childhood cancer survivors: the St Jude Lifetime Cohort Study (SJLIFE), the original and expansion cohorts of the Childhood Cancer Survivor Study (CCSS). The CCSS original cohort had SNP array genotyping and imputation, and the SJLIFE and CCSS expansion cohort underwent whole-genome sequencing. Associations between the variants mapped to GATA3 and risks of different cancer types were analyzed (a specific cancer type was selected as the case group and all other unselected cancer types served as controls, then repeated for 13 cancer types). Summary statistics were subsequently analyzed with the ASSET (Association analysis for SubSETs) method which is powerful for pooling association signals across multiple cancer types when true effects may exist only in a subset and be in opposite directions across cancer types. Results: Genotype data were available for 11623 childhood cancer survivors (5325 from the CCSS original cohort, 2428 from the CCSS expansion cohort, and 3870 from the SJLIFE, all European ancestry) were initially considered. After excluding individuals with dual enrollment or as outliers in the principal components analysis (n=667), a total of 10956 individuals remained for genetic association analysis. We identified a bi-directional association for a subset of childhood cancers with rs3781093 (2-sided P value of 6.22 × 10−19): the same effect allele (C) was associated with increased risk of ALL or soft tissue sarcoma [STS] (OR=1.21; 95% CI=1.13-1.29; P=7.4 × 10−9), and was associated with decreased risk of HL (OR=0.66; 95% CI=0.59-074; P=1.80 × 10−12). The frequencies of the effect allele (C) for rs3781093 in ALL cases (17.9%), STS cases (17.8%) and HL cases (12.5%) relative to non-cancer community controls in the SJLIFE (15.6%) and non-cancer gnomAD samples (14.9%) corroborate this finding. In our combined dataset, there were 1380 Hodgkin lymphoma cases, 2961 ALL cases and 692 STS cases. Conclusion: We discovered a novel variant in the GATA3 locus exhibiting pleiotropy with both tumor-promoting and protective effects, suggesting a shared mechanistic pathway involving GATA3 may underlie the carcinogenesis for ALL, STS and HL. Conducting systematic searches for childhood cancer pleiotropy represents an important research direction. Citation Format: Cheng Chen, Xijun Zhang, Qian Dong, Heather L. Mulder, John Easton, Xiaotu Ma, Jinghui Zhang, Jun Yang, Kim E. Nichols, Gregory T. Armstong, Kirsten K. Ness, Melissa M. Hudson, Hui Wang, Nilanjan Chatterjee, Cindy Im, Zhaoming Wang. Pleiotropic GATA3 locus is associated with multiple childhood cancers: harnessing the existing data from the St. Jude Lifetime Cohort and the Childhood Cancer Survivor Study for genetic etiology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6618.

Clinical Analysis of Philadelphia Chromosome-Like Acute Lymphoblastic Leukemia in Children

To explore the clinical characteristics, molecular characteristics, treatment and prognosis of pediatric Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) with a therapeutic target. A total of 27 patients of Ph-like ALL with targeted drug target were initially diagnosed in Children's Hospital of Soochow University from December 2017 to June 2021. The data of age, gender, white blood cell (WBC) count at initial diagnosis, genetic characteristics, molecular biological changes, chemotherapy regimen, different targeted drugs were given, and minimal residual disease (MRD) on day 19, MRD on day 46, whether hematopoietic stem cell transplantation (HSCT) were retrospective analyed, and the clinical characteristics and treatment effect were summarized. Survival analysis was performed by Kaplan-Meier method. The intensity of chemotherapy was adjusted according to the MRD level during induced remission therapy in 27 patients, 10 patients were treated with targeted drugs during treatment, and 3 patients were bridged with HSCT, 1 patient died and 2 patients survived. Among the 24 patients who did not receive HSCT, 1 patient developed relapse, and achieved complete remission (CR) after treatment with chimeric antigen receptors T cells (CAR-T). The 3-year overall survival, 3-year relapse-free survival and 3-year event-free survival rate of 27 patients were (95.5±4.4)%, (95.0±4.9)% and (90.7±6.3)% respectively. Risk stratification chemotherapy based on MRD monitoring can improve the prognosis of Ph-like ALL in children, combined with targeted drugs can achieve complete remission as soon as possible in children whose chemotherapy response is poor, and sequential CAR-T and HSCT can significantly improve the therapeutic effect of Ph-like ALL in children whose MRD is continuously positive during induced remission therapy.

Acute lymphoblastic leukemia in young adults: which treatment?

Despite improvements in survival among pediatric patients with acute lymphoblastic leukemia (ALL), survival outcomes for adolescents and young adults (AYAs) with ALL have lagged. The reasons for the inferior outcomes among AYAs are multifactorial, each presenting unique challenges and requiring novel solutions. First, adverse disease biology is more common among AYAs with ALL. Ongoing trials are investigating novel approaches to treatment, such as incorporating JAK inhibitors for Philadelphia chromosome-like ALL, menin inhibitors for KMT2A-rearranged ALL, and BCL2/BCLXL inhibition for T-cell ALL. Poorer adherence to therapy also impedes improvements in survival outcomes for AYAs with ALL, but early data suggest that technology, both for monitoring and interventions, may be useful in increasing adherence among this population. Finally, better access to clinical trials and collaboration between pediatric and adult centers is critical in advancing the care of AYAs with ALL. Significant improvements have been made over the past decade, but recognizing, understanding, and addressing each of these unique challenges provides hope that the outcomes for AYAs will continue to improve even further.

Abstract B134: Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate

Abstract Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk hematological malignancy with poor outcome, and new treatments are urgently required. We address this challenge by targeting cytokine receptor-like factor 2 (CRLF2), which is overexpressed in ~50% of Ph-like ALL cases conferring chemoresistance and survival of Ph-like ALL cells through JAK/STAT signaling. CRLF2 is an ideal target due to its role in leukemogenesis, limiting the likelihood of relapse associated with antigen loss, and its minimal expression in healthy tissue. Here, we developed a novel CRLF2-targeting antibody fragment-drug conjugate (CRLF2-FDC) and evaluated its efficacy in preclinical models. Methods: A single-chain anti-CRLF2 variable monoclonal antibody fragment (scFv-Fc) was engineered by conventional hybridoma technology, PCR and expression in HEK293T cells. The scFv-Fc was conjugated to the anti-microtubule drug DM1 via a non-cleavable linker. The selective association and internalization of the CRLF2-FDC was determined in vitrousing Ph-like ALL cell lines and their CRLF2-knockdown counterparts, and ex vivo using patient-derived xenograft (PDX) cells. The in vivo efficacy of the CRLF2-FDC (equivalent to 10 mg/kg scFv-Fc, 0.4 mg/kg DM1, once weekly x 4 weeks via intravenous injection) was tested against pediatric ALL PDX models in immune-deficient (NSG) mice, in comparison to scFv-Fc or DM1 alone. The efficacy of the CRLF2-FDC was further evaluated following two weeks of standard-of-care induction chemotherapy [vincristine (0.15 mg/kg, once weekly), dexamethasone (5 mg/kg) and L-asparaginase (1,000 U/kg) both 5 days on 2 days off (VXL)]. Treatment efficiency was calculated by subtracting the median event-free survival (EFS) of the control and treated groups (T-C) and stringent objective response criteria (progressive disease, PD; partial response, PR; complete response, CR; maintained CR, MCR) and leukemia infiltration in major organs at day 28 post treatment initiation. Results: CRLF2-dependent association (P≤0.01) and internalization of the CRLF2-FDC was confirmed both in vitro and in vivo in Ph-like ALL cells. While the scFv-Fc alone or free DM1 elicited PDs in a Ph-like ALL PDX in vivo (T-C 6 and 1.6 days, respectively, P=0.0013 for both), the CRLF2-FDC achieved a PR (T-C 32 days, P=0.0088). Besides targeted drug delivery, the CRLF2-FDC also demonstrated a significant antagonistic impact on STAT5 signaling in vivo 8 h after administration, suggesting a two-pronged therapeutic approach. Notably, the CRLF2-FDC achieved an MCR when administered after VXL (T-C 53.9 days, P <0.0001) compared to a CR (T-C 25.2 days, P <0.0001) by VXL alone. Furthermore, the CRLF2-FDC significantly decreased leukemic burden in major organs at 28 days post treatment initiation. Conclusions: This study describes the selective potency and promising preclinical activity of a novel CRLF2-FDC for the treatment of Ph-like ALL when administered either as a single agent or following standard-of-care induction therapy. Citation Format: Richard B Lock, Sara M.A. Mohamed, Peter Schofield, Karl-Heinz Friedrich, Daniel Christ, Maria Kavallaris, Narges Bayat. Targeting Philadelphia chromosome-like acute lymphoblastic leukemia with a novel CRLF2 antibody fragment-drug conjugate [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B134.

Real World Outcomes in Patients with Ph-like ALL at the University of New Mexico

Introduction: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a heterogenous subtype of ALL characterized by different translocations and molecular mutations, resulting in a gene expression profile similar to Philadelphia chromosome positive (Ph+) ALL without an identified BCR::ABL1 translocation. Unfortunately, Ph-like ALL is characterized by worse response rates to conventional therapies and poor overall survival compared to other ALL subtypes. It is reported that 20-30% of patients with newly diagnosed ALL in the United States have Ph-like ALL and the incidence appears to vary according to ethnicity. While many therapies are under investigation including the addition of a tyrosine kinase inhibitor or ruxolitinib to conventional chemotherapy, a standardized and molecularly-based approach to treating patients with Ph-like ALL is not yet defined. There also remain questions about the efficacy of integrating inotuzumab ozogamicin or blinatumomab into first-line therapy for these patients. In addition, recent retrospective data has demonstrated promising effects of using traditional salvage therapies for ALL such as Car T cells, inotuzumab, or blinatumomab in patients with Ph-like ALL. However, the optimal sequence of treatment as well the efficacy of combining salvage therapies with agents such as ruxolitinib remains unknown. Herein, we present our institutional experience with diagnosing and treating adult patients with Ph-like ALL at the University of New Mexico. Our goal was to characterize the demographics, molecular and cytogenetic landscape, and treatment outcomes at a medical center that serves a patient population enriched for this disease. Methods: After IRB approval, we performed a retrospective chart review of patients diagnosed and treated at the University of New Mexico between January 1, 2018 and April 1, 2023. We included patients who were 18 years or older at the time of diagnosis, received first-line therapy at the University of New Mexico, and had gene expression profiling or FISH testing that was consistent with Ph-like ALL. Statistical analysis was performed using SPSS Statistics v.27 (IBM, 2020). Results: We identified 9 patients who met inclusion criteria. All 9 patients self-identified as either ‘American Indian or Alaska Native’ or ‘Hispanic or Latino.‘ The median age at diagnosis was 34 and the mean BMI at diagnosis was 34.6. 5 patients were CD20 positive and 1 patient had a concurrent IKZF1 mutation. All patients received induction chemotherapy with either a BFM regimen or HyperCVAD. 5 (55%) patients were refractory to induction and 6 (66%) patients eventually underwent allogeneic stem cell transplant as part of their treatment. 1 patient received Car T cell therapy with Brexucabtagene autoleucel following relapse after allogeneic stem cell transplant. 1 patient died 124 days after diagnosis and 8 patients are alive currently. 4 patients had IGH:: CRLF2, 2 patients had P2RY8::CRLF2,1 patient had GTF2I::PDGFRB,1 patient had IGH::BCL2,and one patient had both a positive low-dose array and a positive gene expression profile without an identified molecular or cytogenetic abnormality consistent with Ph-like ALL. Discussion: We present our preliminary experience of patients with Ph-like ALL diagnosed and treated at the University of New Mexico. Our patients were young with a median age of 34. Consistent with published data describing patients with Ph-like ALL, our patients had poor responses to traditional ALL treatments and most commonly had an identified fusion with CLRF2. Our data supports the urgent need for the development of further treatment options for patients with Ph-like ALL as well as clinical trials aimed at standardizing management.

Synergy of Ruxolitinib and Carfilzomib in Targeting the PAX5::JAK2 fusion I n Vitro: Potential Therapeutic Advantage for a Subset of Ph-like Acute Lymphoblastic Leukemia

INTRODUCTION PAX5::JAK2 is a gene fusion that drives a subset of Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) and confers aggressive disease, poor prognosis and poor response to treatment. PAX5::JAK2 is present in approximately 12% of Ph-like ALL cases. Targeted therapy is urgently required to improve outcomes. We evaluated the efficacy of JAK inhibitor ruxolitinib (RUX) and proteosome inhibitors carfilzomib (CFZ) and bortezomib (BTZ) as monotherapy and in combination against PAX5::JAK2 transfected Ba/F3 cells in vitro. Downregulation of proteosome genes via reduction in STAT3 phosphorylation is a shared mechanism of RUX and proteosome inhibitors. Given that the PAX5::JAK2 fusion results in increased STAT3 phosphorylation driving proliferation, it was proposed that the combination of proteosome inhibitors and RUX would demonstrate synergy in inhibiting proliferation of PAX5::JAK2 driven leukemic cells. METHODS Mouse derived Ba/F3 cells, an IL-3 dependent B-ALL model, were transfected with the PAX5::JAK2 fusion and demonstrated IL-3 independence. We applied, as single treatments, differing concentrations of RUX and the proteosome inhibitors CFZ and BTZ to PAX5::JAK2 transfected Ba/F3 cells and two control cell lines - IL-3 dependent empty vector Ba/F3 cells and an IL-3 independent KG1a myeloid cell line. Differing concentrations of CFZ and 200nM of RUX were then applied in combination. 200nM of RUX was selected because it is a dose at which the drug has minimal effect as a single agent. Inhibitors were applied to cells for 72 hours then percentage of live and dead cells at each concentration was ascertained by flow cytometry using Annexin V and LIVE/DEAD fixable aqua dead cell stain. Downstream STAT5 and STAT3 activation in basal state, and as a response to treatment, was assessed using phosphoflow. We demonstrated mechanisms of growth inhibition via reduction in STAT5 and STAT3 phosphorylation by the inhibitors. RESULTS RUX, CFZ and BTZ were all effective against PAX5::JAK2 transfected Ba/F3 cells with a median lethal dose (LD50) of 514nM (CI 498-530nM) for RUX, 38nM (CI 36-39nM) for CFZ and 19nM (CI 18-20nM) for BTZ. RUX had a variable effect on IL-3 dependent empty vector control cells which also demonstrate JAK/STAT activation, but no effect on the IL-3 independent KG1a cells. CFZ had a lesser effect on empty vector control cells with a LD50 of 59nM (CI 58-60nM) and no effect on KG1a control cells. BTZ had a significant effect on both control cell lines, predicting off-target toxicity and consequently was not applied in combination with RUX. RUX and CFZ in combination demonstrated an LD50 of 21nM (CI 18-25nM) (see figure 1) which is significantly lower than the LD50s for either agent used as monotherapy. Combination index for RUX and CFZ at 45nM was 0.80 and at 50nM was 0.49, suggesting moderate synergy. Phosphoflow demonstrated reduced pSTAT5 with the application of RUX and CFZ to PAX5::JAK2 transfected Ba/F3 cells with mean fluorescence intensity (MFI) reducing from 351 to 285 in inhibitor treated cells, while for STAT3 MFI reduced from 396 to 249. (see figure 2) There was no effect on MFI for control cell lines. This demonstrates that the mechanism for growth inhibition is inhibition of JAK/STAT signaling due to the reduction in pSTAT3 and pSTAT5. CONCLUSIONS We showed in vitro that RUX, CFZ and BTZ are all potent against the high risk PAX5::JAK2 fusion. RUX and CFZ in combination demonstrated moderate synergy against PAX5::JAK2 and inhibited proliferation through their effect on JAK/STAT pathways. The RUX/CFZ combination, drugs widely available in clinical practice, is particularly promising given the apparent synergistic effect on limiting leukemic cell growth. Such treatments warrant clinical trials for management of the poor prognostic group of Ph-like ALL with PAX5::JAK2, given that they may enhance efficacy and lessen toxicity. Clinical trials have already demonstrated efficacy and safety of RUX and CFZ as single agents in combination with chemotherapy for the treatment of B-ALL which indicates the feasibility of combining the two agents clinically in Ph-like ALL.

Prognostic analysis of children with Philadelphia chromosome-like acute lymphoblastic leukemia common genes

Objective: To summarize the clinical data and prognosis of children with Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) common genes. Methods: This was a retrospective cohort study.Clinical data of 56 children with Ph-like ALL common gene cases (Ph-like ALL positive group) treated from January 2017 to January 2022 in the First Affiliated Hospital of Zhengzhou University, Henan Children's Hospital, Henan Cancer's Hospital and Henan Provincial People's Hospital were collected, 69 children with other high-risk B cell acute lymphoblastic leukemia (B-ALL) at the same time and the same age were selected as the negative group. The clinical characteristics and prognosis of two groups were analyzed retrospectively. Comparisons between groups were performed using Mann-Whitney U test and χ2 test. Kaplan-Meier method was used for survival curve, Log-Rank test was used for univariate analysis, and the Cox regression model was used for multivariate prognosis analysis. Results: Among 56 Ph-like ALL positive patients, there were 30 males and 26 females, and 15 cases were over 10 years old. There were 69 patients in Ph-like ALL negative group. Compared with the negative group, the children in positive group were older (6.4 (4.2, 11.2) vs. 4.7 (2.8, 8.4) years), and hyperleukocytosis (≥50×109/L) was more common (25% (14/56) vs. 9% (6/69)), the differences were statistically significant (both P<0.05). In the Ph-like ALL positive group, 32 cases were positive for IK6 (1 case was co-expressed with IK6 and EBF1-PDGFRB), 24 cases were IK6-negative, of which 9 cases were CRLF2 positive (including 2 cases with P2RY8-CRLF2, 7 cases with CRLF2 high expression), 5 cases were PDGFRB rearrangement, 4 cases were ABL1 rearrangement, 4 cases were JAK2 rearrangement, 1 case was ABL2 rearrangement and 1 case was EPOR rearrangement. The follow-up time of Ph-like ALL positive group was 22 (12, 40) months, and 32 (20, 45) months for negative group. The 3-year overall survival (OS) rate of positive group was significantly lower than the negative group ((72±7) % vs. (86±5) %, χ2=4.59, P<0.05). Compared with the 24 IK6-negative patients, the 3-year event free survival (EFS) rate of 32 IK6 positive patients was higher, the difference was statistically significant ((88±9) % vs. (65±14) %, χ2=5.37, P<0.05). Multivariate Cox regression analysis showed that the bone marrow minimal residual disease (MRD) not turning negative at the end of first induction (HR=4.12, 95%CI 1.13-15.03) independent prognostic risk factor for patient with Ph-like ALL common genes. Conclusions: Children with Ph-like ALL common genes were older than other high-risk B-ALL patients at diagnosis, with high white blood cells and lower survival rate. The bone marrow MRD not turning negative at the end of first induction were independent prognostic risk factor for children with Ph-like ALL common gene.

A Phase 2 Study of Ruxolitinib with Chemotherapy in Children with Philadelphia Chromosome-like Acute Lymphoblastic Leukemia (AALL1521/INCB18424-269): Biologic Characteristics and Minimal Residual Disease Response of Patients with Non-CRLF2-Rearranged JAK Pathway Alterations

A Phase 2 Study of Ruxolitinib with Chemotherapy in Children with Philadelphia Chromosome-like Acute Lymphoblastic Leukemia (AALL1521/INCB18424-269): Biologic Characteristics and Minimal Residual Disease Response of Patients with Non-<i>CRLF2</i>-Rearranged JAK Pathway Alterations

Transcriptional Rewiring and Therapeutic Resistance in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Transcriptional Rewiring and Therapeutic Resistance in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

A Clinical Laboratory-Oriented Targeted RNA-Seq System Accurately and Efficiently Detected Various Fusion Gene Mutations in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Introduction: Genetic mutations that occur in blood cancer can be broadly classified into nucleotide sequence mutations including duplications or deletions, fusion gene mutations, and abnormal gene overexpression. Among these genetic mutations, gene fusion not only plays an important role in the development of blood cancers, such as leukemia, but is also important as an essential marker for the diagnosis, risk assessment, optimal treatment selection, and residual lesion detection. Most clinical laboratories use conventional cytogenetic testing, fluorescence in situ hybridization, and multiplex reverse transcriptase-polymerase chain reaction (PCR) to detect fusion gene mutations. However, the currently used methods have great limitations in detecting various gene fusions occurring in blood cancer. To overcome these shortcomings, RNA sequencing (RNA-seq) methods are being introduced in clinical laboratories. The authors developed a targeted RNA-seq panel, a bioinformatics pipeline used for targeted RNA-seq to detect gene fusions, sequence variants, and altered gene expression, which can be used more conveniently in clinical laboratories. Methods: The targeted RNA-seq panel and analysis pipeline developed based on information technology with an automated reporting system and commercialized by the authors was applied to a comparative evaluation with the commercialized targeted RNA-seq panel (Archer™ FusionPlex™ Heme Panel version 2; Archer DX, Boulder, CA, USA) using samples of acute leukemia patients. A laboratory-oriented targeted RNA-seq system consisting of 84 genes was developed in consideration of the test reporting time and operational efficiency in clinical laboratories (KBB-RNAseq NGS-Leukemia-PHB; KBlueBio Inc., Hwasun, Korea). For comparison with the existing analysis system, the Archer FusionPlex Heme v2 panel (ArcherDx) based on the cDNA library for specific genes using anchored multiplex PCR was used. To evaluate the test performance of these systems, bone marrow specimens were used while diagnosing 93 patients with leukemia (15 cases of acute myeloid leukemia, 35 cases of adult B-acute lymphoid leukemia, 30 cases of childhood B-acute leukemia, and 13 cases of T-acute lymphoid leukemia). Results: Among all 93 leukemia patients, tier 1 or tier 2 gene fusions were observed in 72 (77%) patients. Gene fusions were detected in 83% (25/30) of patients with pediatric B-acute lymphoid leukemia (B-ALL), and in 94% (33/35) of patients with adult B-ALL. In patients with acute myeloid leukemia (AML) and T-ALL, the proportions of fusion gene mutations were 53% (8/15) and 46% (6/13), respectively. For the comparative evaluation, 4 cases of B-ALL, 2 cases of AML, and 1 case of acute promyelocytic leukemia and T-ALL were used. The results of the two analysis systems were consistent in 7 of the 8 comparatively evaluated patients, but an IGH-CRLF2 fusion in a B-ALL case was detected only with the panel developed by the authors. It is known that the CRLF2 gene is a causative gene in half of the Philadelphia chromosome (Ph)-ALL cases, which account for 20-25% of all B-ALL cases and are classified as a new type of ALL showing a very poor prognosis. Conclusions: Our targeted RNA-seq system accurately and efficiently detected various fusion gene mutations that occur in acute leukemia. Importantly, our targeted RNAseq panel detected fusion gene mutations that cause Ph-like B-ALL more accurately than the c-ommercialized test system. This targeted RNA-seq system showed a reliable and stable performance in detecting various gene fusions occurring in blood cancers, such as leukemia, in clinical laboratories. Also, our targeted RNA-seq system was found to be very useful in diagnosing Ph-like B-ALL compared to the commercial RNA-seq panel. Keywords: Targeted RNA-seq, Clinical Laboratory, Ph-like B-ALL, Acute leukemia Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BCL6 inhibition ameliorates resistance to ruxolitinib in CRLF2-rearranged acute lymphoblastic leukemia.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is an intractable disease and most cases harbor genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits a CRLF2 gene rearrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient as a treatment, so combinatorial therapies targeting multiple signals are needed. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, we explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and found that elevated BCL6 expression was one such mechanism. Upregulated BCL6 suppressed the expression of TP53 along with its downstream cell cycle inhibitor p21 (CDKN2A) and pro-apoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells some degree of resistance to therapy. BCL6 inhibition (with FX1) alone was able to upregulate TP53 and restore the TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis- sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged the survival of xenografted mice. These findings provide one mechanism for the insufficiency of JAK inhibition for the treatment of CRLF2-rearranged ALL and indicate BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Sequential Approach to Improve the Molecular Classification of Childhood Acute Lymphoblastic Leukemia

Identification of specific leukemia subtypes is a key to successful risk-directed therapy in childhood acute lymphoblastic leukemia (ALL). Although RNA sequencing (RNA-seq) is the best approach to identify virtually all specific leukemia subtypes, the routine use of this method is too costly for patients in resource-limited countries. This study enrolled 295 patients with pediatric ALL from 2010 to 2020. Routine screening could identify major cytogenetic alterations in approximately 69% of B-cell ALL (B-ALL) cases by RT-PCR, DNA index, and multiplex ligation-dependent probe amplification. STIL-TAL1 was present in 33% of T-cell ALL (T-ALL) cases. The remaining samples were submitted for RNA-seq. More than 96% of B-ALL cases and 74% of T-ALL cases could be identified based on the current molecular classification using this sequential approach. Patients with Philadelphia chromosome-like ALL constituted only 2.4% of the entire cohort, a rate even lower than those with ZNF384-rearranged (4.8%), DUX4-rearranged (6%), and Philadelphia chromosome-positive (4.4%) ALL. Patients with ETV6-RUNX1, high hyperdiploidy, PAX5 alteration, and DUX4 rearrangement had favorable prognosis, whereas those with hypodiploid and KMT2A and MEF2D rearrangement ALL had unfavorable outcomes. With the use of multiplex ligation-dependent probe amplification, DNA index, and RT-PCR in B-ALL and RT-PCR in T-ALL followed by RNA-seq, childhood ALL can be better classified to improve clinical assessments.

The combination of ruxolitinib and Bcl-2/Mcl-1 inhibitors has a synergistic effect on leukemic cells carrying a SPAG9::JAK2 fusion.

JAK2 rearrangements can occur in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). Here, we performed functional analysis of the SPAG9::JAK2 fusion, which was identified in a pediatric patient with Ph-like ALL, to establish molecular targeted therapy. Ba/F3 cells expressing SPAG9::JAK2 generated by retroviral transduction (Ba/F3-SPAG9-JAK2), proliferated in the absence of IL-3, and exhibited constitutive phosphorylation of the tyrosine residues in the JAK2 kinase domain of the fusion protein and STAT3/STAT5. Mutation of tyrosine residues in the JAK2 kinase domain (SPAG9::JAK2 mut) abolished IL-3 independence, but had no influence on STAT3/STAT5 phosphorylation levels. Gene expression analysis revealed that Stat1 was significantly upregulated in Ba/F3-SPAG9-JAK2 cells. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells, suggesting that STAT1 is key for SPAG9::JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9::JAK2, but not SPAG9::JAK2 mut. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro, but was insufficient in vivo. Venetoclax (a BCL-2 inhibitor) or AZD5991 (an MCL-1 inhibitor) enhanced the effects of ruxolitinib on Ba/F3-SPAG9-JAK2 in vitro. These findings suggest that activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to SPAG9::JAK2-related aberrant growth promotion. BCL-2 or MCL-1 inhibition is a potential therapeutic option for B-ALL with SPAG9::JAK2 fusion.

Treatments of Ph-like acute lymphoblastic leukemia: a real-world retrospective analysis from a single large center in China

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL. We retrospectively studied 70 cases with Ph-like ALL and here present the largest study of CAR-T cell treatment and haplo-HSCT for this leukemia. Median age was 26 years and median leukocyte count was 31.44 × 109/L. The proportion of patients receiving chemotherapy, KIs, CAR-T cells, and allo-HSCT was 19%, 30%, 46%, and 61%, respectively. The overall response rate was 62%, 73%, and 100% after one month of KI treatment combined with chemotherapy, CAR-T cell therapy, and allo-HSCT, respectively. Five-year DFS and OS were 35% and 51%, respectively. The five-year cumulative incidence of relapse and non-relapse mortality was 63% and 11%, respectively. Allo-HSCT was associated with a better DFS (p = 0.010) and OS (p = 0.000) by univariate analysis. In conclusion, allo-HSCT after KIs together with chemotherapy or CAR-T cell therapy is a safe and feasible treatment modality for Ph-like ALL.

Genomic profile of radiation-induced early-onset mouse B-cell lymphoma recapitulates features of Philadelphia chromosome-like acute lymphoblastic leukemia in humans.

Epidemiological studies have revealed a radiation-related increase in the risk of developing acute lymphoblastic leukemia (ALL). Our recent study revealed early induction and increased risk of precursor B-cell (pB) lymphomas in mice after radiation exposure. However, the genomic landscape of radiation-induced B-cell lymphomas remains unclear. To identify the relevant genetic alterations in mice, whole-exome sequencing was performed on both early-onset and late-onset B-cell lymphomas that developed spontaneously or after gamma-irradiation. In addition to multiple driver mutations, the data revealed that interstitial deletion of chromosome 4, including Pax5, and missense mutations in Jak3 are unique genomic alterations in radiation-induced, early-onset B-cell lymphomas. RNA sequencing revealed a pB-cell-type gene-expression profile with no involvement of known fusion genes for human ALLs in the early-onset B-cell lymphomas. Activation of Jak3/Stat5 signaling in early-onset B-cell lymphomas was validated using western capillary electrophoresis. Those features were similar to those of Philadelphia chromosome-like ALL. Our data suggest a critical role for Pax5 loss-of-function mutations in initiating B-cell leukemogenesis coupled with activation of Jak3/Stat5 signaling as a basis for the rapid development of radiation-induced pB-ALL. These molecular signatures for radiation-induced cancers will inform both risk assessment and potential targeted therapies for pB-ALL.

Noncoding genetic variation in GATA3 increases acute lymphoblastic leukemia risk through local and global changes in chromatin conformation.

Inherited noncoding genetic variants confer significant disease susceptibility to childhood acute lymphoblastic leukemia (ALL) but the molecular processes linking germline polymorphisms with somatic lesions in this cancer are poorly understood. Through targeted sequencing in 5,008 patients, we identified a key regulatory germline variant in GATA3 associated with Philadelphia chromosome-like ALL (Ph-like ALL). Using CRISPR-Cas9 editing and samples from patients with Ph-like ALL, we showed that this variant activated a strong enhancer that upregulated GATA3 transcription. This, in turn, reshaped global chromatin accessibility and three-dimensional genome organization, including regions proximal to the ALL oncogene CRLF2. Finally, we showed that GATA3 directly regulated CRLF2 and potentiated the JAK-STAT oncogenic effects during leukemogenesis. Taken together, we provide evidence for a distinct mechanism by which a germline noncoding variant contributes to oncogene activation, epigenetic regulation and three-dimensional genome reprogramming.

Precision Co-Targeting of the Thymic Stromal Lymphopoietin Receptor in Childhood CRLF2-Rearranged Acute Lymphoblastic Leukemia

Introduction : Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is associated with high rates of chemoresistance and relapse. CRLF2 (cytokine receptor-like factor 2) rearrangements occur in 50% of Ph-like and 60% of Down Syndrome (DS)-associated ALL and induce constitutive JAK/STAT and other kinase signaling. Current clinical trials are studying chemotherapy with the JAK inhibitor ruxolitinib in patients with CRLF2-rearranged Ph-like ALL, but results are not yet known. While chimeric antigen receptor T-cell (CART) immunotherapies have induced remarkable remissions in children with relapsed/refractory B-ALL, approximately 50% of CD19CART-treated patients will relapse again, many with CD19 antigen loss. New therapies are needed to prevent relapse and overcome immunotherapeutic resistance.Methods : We previously developed CAR T cells targeting the thymic stromal lymphopoietin receptor (TSLPR; encoded by CRLF2) and demonstrated potent preclinical activity in Ph-like ALL models (Qin Blood 2015), which has led to a soon-to-open phase 1 clinical trial for patients with relapsed/refractory CRLF2-overexpressing ALL. In the current preclinical studies, we hypothesized that combinatorial targeting with bispecific TSLPRxCD19CART or TSLPRxCD22CART (Ross Cancer Res 2020) or with TSLPRCART + ruxolitinib will have superior activity against CRLF2-rearranged Ph-like and DS-ALL.Results : TSLPRCART treatment of CRLF2-rearranged ALL cell line (n=1) and patient-derived xenograft (PDX) models potently inhibited leukemia proliferation in vitro and in vivo and induced long-term ‘cure’ of xenograft mice. However, co-administration of TSLPRCART + ruxolitinib markedly diminished in vivo T cell numbers, blunted cytokine production, and facilitated leukemia relapse, which could be abrogated by delaying ruxolitinib. Importantly, ruxolitinib co-treatment prevented severe TSLPRCART-induced cytokine release syndrome (CRS) and animal death. Interestingly, ruxolitinib withdrawal led to return of T-cell functionality with re-detection of TSLPRCART in peripheral blood, induction of IFN-γ production, and leukemia clearance upon CRLF2+ ALL rechallenge (Figure 1).Conclusions: In these preclinical studies, we report potent activity of TSLPRCART in cell line (n=1) and PDX models of childhood CRLF2-rearranged Ph-like ALL (n=2) and DS-ALL (n=2) and, interestingly, deleterious effects of concomitant JAK inhibition upon CAR T cell functionality. We demonstrated that ruxolitinib co-administration impaired in vivo TSLPRCART-induced ALL cell killing but was also beneficial in protection against life-threatening cytokine release syndrome in co-treated animals. Importantly, TSLPRCART was not eliminated, only suppressed, by JAKi co-treatment with restoration of T cell functionality upon ruxolitinib removal and/or leukemia relapse/rechallenge studies. Ongoing studies are defining optimal TSLPRCART + ruxolitinib sequence(s) to maximize both anti-leukemia efficacy and potential CRS mitigation, as well as assessing in vivo efficacy of bispecific TSLPRCARTs in CRLF2-R Ph-like ALL and DS-ALL PDX models for future translation and clinical evaluation in next-generation trials. [Display omitted] DisclosuresFry: ElevateBio: Research Funding; Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tasian: Aleta Biotherapeutics: Consultancy; Kura Oncology: Consultancy; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Activation of the STAT1-BCL-2/MCL-1 Axis in Leukemic Cells Carrying a SPAG9-JAK2 Fusion

[Background and aim of this study]Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a distinct subtype of B-ALL with poor prognosis. JAK2 related fusion genes have been identified in this subtype, especially in adolescent and young adults (AYA). Previously, we identified SPAG9-JAK2 fusion gene in 14-year-old boy with Ph-like ALL (Kawamura M, et al. Genes Chromosomes Cancer. 2015). In this study, we performed functional analysis of the SPAG9-JAK2 fusion protein, and evaluated the efficacy of treatment with a JAK inhibitor against cells carrying the fusion. In addition, we assessed therapeutic options other than JAK2 inhibition following comprehensive molecular analysis.[Materials and Methods]Full length of SPAG9-JAK2 cDNA was cloned into retroviral construct with Tet-On system. Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing SPAG9-JAK2 (Ba/F3-SPAG9-JAK2) under doxycycline (DOX) dependent manner. Ba/F3-SPAG9-JAK2 were analyzed whether IL-3 independent growth was achieved. Aberrant activation of JAK-STAT pathway achieved by SPAG9-JAK2 was evaluated by western blot. To clarify whether the tyrosine residues of JAK2 in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing SPAG9-JAK2 mutants (SPAG9-JAK2 mut) in which both tyrosine residues of JAK2 were replaced with phenylalanine was established. Gene expression analysis using Mouse Genome 430 2.0 Array was performed for comprehensive analysis of gene expression profile related to SPAG9-JAK2. Sensitivity of Ba/F3-SPAG9-JAK2 to ruxolitinib (a JAK inhibitor) was tested in cytotoxic assay and in xenograft model established using Ba/F3-SPAG9-JAK2 cells.[Results and discussions]The expression of SPAG9-JAK2 in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-SPAG9-JAK2 proliferated without IL-3 in contrast to Ba/F3 cells (p<0.01), suggesting SPAG9-JAK2 had the proliferation activity. Western blot revealed that constitutive phosphorylation of tyrosine residue of SPAG9-JAK2, STAT3/STAT5, suggesting constitutive activation of JAK2-STAT3/STAT5 pathway. SPAG9-JAK2 mut abolished IL-3 independence (p<0.01), but had no influence on STAT3/STAT5 phosphorylation levels detected by western blot. Gene expression analysis revealed that Stat1 was significantly up-regulated in Ba/F3-SPAG9-JAK2 cells compared with mock Ba/F3 cells (fold change 8.04 with p < 0.01) [Fig. 1], confirmed by western blot. STAT1 was also phosphorylated in Ba/F3-SPAG9-JAK2 but not SPAG9-JAK2 mut cells detected by western blot [Fig. 2], suggesting that STAT1 is a key mediator for SPAG9-JAK2-mediated cell proliferation. Consistently, STAT1 induced expression of the anti-apoptotic proteins, BCL-2 and MCL-1, as did SPAG9-JAK2, but not SPAG9-JAK2 mut confirmed by western blot [Fig. 3]. Ruxolitinib abrogated Ba/F3-SPAG9-JAK2-mediated proliferation in vitro (p<0.01), with an 50% inhibitory concentration (IC50) value of 65.9 ± 9.8 nM, causing decrease of JAK2, STAT1/STAT3/STAT5 phospholyration in western blot and apoptosis in annexin V/PI staining. Ruxolitinib prolonged survival time of xenotransplanted mice (p = 0.0213), however, the proliferation of leukemic cells in mouse bone marrow was not suppressed by ruxolitinib. Ba/F3-SPAG9-JAK2 cells showed a dose-dependent response for venetoclax (a BCL-2 inhibitor) with IC50 2.57 ± 1.11 µM and AZD5991 (an MCL-1 inhibitor) with IC50 6.76 ± 3.31 µM. Treatment of Ba/F3-SPAG9-JAK2 cells with a combination of ruxolitinib and venetoclax or AZD5991 resulted in a significant reduction in the IC50 of ruxolitinib (p<0.01) [Fig. 4, 5], with a combined index (CI) value of 0.61 or 0.92, indicating a moderately or weak synergistic effect in vitro.[Conclusion]SPAG9-JAK2 promotes cell proliferation and that tyrosine phosphorylation of the JAK2 kinase domain is critical for IL-3-independent cell growth. Ruxolitinib shows sufficient cytotoxic effects against Ba/F3-SPAG9-JAK2 cells in vitro, but is only partially effective in vivo. Activation of the JAK2-STAT1-BCL-2/MCL-1 axis contributes to aberrant growth promotion by SPAG9-JAK2. BCL-2 or MCL-1 inhibitors in combination with ruxolitinib shows efficacy against Ph-like ALL carrying the SPAG9-JAK2 fusion in vitro. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide CRISPR-Cas9 Screen Identifies Rationally Designed Combination Therapies Relevant for CRLF2-Rearranged Ph-like ALL

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL, also known as BCR-ABL1-like ALL) is a disease entity of B-cell ALL that exhibits a gene expression profile, similar to that of Philadelphia chromosome-positive ALL. Ph-like ALL is categorized into two disease subtypes: “ABL-class”- and “CRLF2/JAK pathway”-types, both of which harbor gene alterations that constitutively activate cytokine/growth factor-related signals. Ph-like ALL with the CRLF2 rearrangement exhibits poor clinical outcomes and is the most common subtype of Ph-like ALL. Tyrosine kinase inhibitor (TKI)-based treatment regimens are effective in treating ABL-class type Ph-like ALL; however, no standard regimen has been established for the CRLF2/JAK pathway type. While multiple chemotherapeutic regimens, including Ruxolitinib monotherapy and/or its combination with chemotherapy, are being tested, their efficacy is reportedly limited. Thus, novel approaches are needed to treat CRLF2/JAK pathway type Ph-like ALL, in particular for CRLF2-rearranged (CRLF2-r) ALL.To identify molecules/pathways relevant for CRLF2-r ALL pathogenesis, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of Ruxolitinib using two IgH-CRLF2-r ALL lines (MUTZ5 and KOPN49) that differ in RAS mutational status. To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which, for the first time, enabled highly efficient transduction of human B cell amenable for genome-wide CRSPR/Cas9 screens.While sgRNAs targeting CRLF2, IL7RA or JAK1/2 significantly affected cell fitness in both lines, those targeting STAT5A, STAT5B or STAT3 did not, suggesting that the JAK-STAT axis is largely dispensable for IgH-CRLF2-r ALL cell survival. We show that regulators of RAS signaling are critical for cell fitness and Ruxolitinib sensitivity and that CRKL and FLT3 depletion enhances Ruxolitinib sensitivity in RAS wild-type (WT) cells. Gilteritinib, a pan-tyrosine kinase inhibitor that reduces CRKL activity, effectively killed RAS WT IgH-CRLF2-r ALL cells in vitro and in vivo, either alone or combined with Ruxolitinib. We further show that combining Gilteritinib with Trametinib, a MEK1/2 inhibitor, is an effective means to target IgH-CRLF2-r ALL cells regardless of RAS mutational status.Our study delineates molecules/pathways relevant for CRLF2-r ALL pathogenesis and could suggest rationally designed combination therapies appropriate for disease subtypes. DisclosuresNo relevant conflicts of interest to declare.

Outcomes of Allogeneic Hematopoietic Cell Transplantation in Adults with Ph-like ALL

Outcomes of Allogeneic Hematopoietic Cell Transplantation in Adults with Ph-like ALL