Lineage Switch Research Articles

Lineage Switching Occurs Upon Relapse in Pediatric Acute Myeloid Leukemia.

Relapsed tumors in pediatric AML are enriched in primitive cells and lose myeloid transcriptional programs.

Project Evolve, Evaluation of Lineage Switch (LS), an International Initiative: Preliminary Results Reveal Dismal Outcomes in Patients with LS

Introduction: Lineage switch (LS) refers to the transformation of acute leukemia from one cell lineage to another (e.g., lymphoid (ALL) to myeloid (AML)). This rare phenomenon, distinct from treatment induced malignancy (i.e., t-AML) and mixed phenotype acute leukemia (MPAL), is increasingly being observed following cell surface antigen targeting. Given challenges in the diagnosis and treatment of LS, Project EVOLVE was developed to globally identify cases of LS following immunotherapy. Methods: A protocol and data collection form for this IRB exempt study was widely disseminated to capture deidentified LS case information across multiple centers and cancer consortia. This included collection of data from published reports of LS, with authors contacted to obtain additional information. For this analysis, LS was defined by the emergence of cell-surface markers warranting reclassification of the original leukemia as a different lineage derivation based on the immunophenotype, including cases of AML emerging alongside the original ALL. “Confirmed” LS cases included those where retention of original cytogenetic or molecular aberrations and/or the clonal immunoglobulin rearrangement patterns were verified to confirm the clonal relationship. “Suspected” LS cases included those where samples to confirm clonality marker retention could not be obtained, but the clinical presentation was consistent with LS. The term “transition” was applied when immunophenotypic shifts were transient. Cases with new cytogenetic abnormalities unrelated to the original diagnosis and compatible with t-AML were excluded. Statistical analysis was primarily descriptive. Data cut-off was July 26, 2023. Results: A total of 58 cases were identified, including 19 cases referenced in prior publications. Of the 58, 11 cases were excluded from this initial analysis, including: 4 cases of t-AML, 2 cases of T-ALL to AML, and 5 “transition” cases. Collectively, 47 cases of LS (43 “confirmed”; 4 “suspected”), spanning across 3 continents and 8 countries, were analyzed. (Fig. 1A) Cases included transition from B-ALL to AML in 36 (76.6%) and to MPAL/biphenotypic/ambiguous lineage in 11 (23.4%). Two likely had MPAL at diagnosis and a third with chronic myeloid leukemia. The median age at initial diagnosis was 8.4 years (range, 1 day-76.5 years); median age at LS presentation was 11.0 years (range 0.4-77.3 years). Twenty-three (48.9%) were male and 19 (40.4%) had prior stem cell transplant (SCT). Baseline cytogenetics showed KMT2A rearrangement in 27 (57.4%). Baseline myeloid antigen co-expression was seen in 22 of 23 patients with submitted data, but variation in degree of expression across patients and antigens was substantial. The most proximal immunotherapy prior to development of LS was CAR T-cells in 23 (48.9%) and blinatumomab in 24 (51.1%). The median time from the most proximal immunotherapy to development of LS was 1.6 months (range, 7 days-36.5 months). In 7 (29.2%) patients receiving blinatumomab, LS developed during the infusion. LS presented as isolated marrow relapse in 27 (57.4%), isolated CNS in 2 (4.3%), isolated non-CNS extramedullary disease in 3 (6.4%), combined relapse in 14 (29.8%) and unreported in 1. First line treatment for LS was chemotherapy induction in 31 (66.0%), palliative care/no therapy in 8 (17.0%), and alternative therapies in the remaining 8 (17.0%). In 35 (74.5%) patients, remission induction was the goal of first line therapy. Fifteen (31.9%) achieved a complete remission (CR) of whom 2 were treated with palliative intent. Across all patients, 8 (17.0%) are alive in remission, the longest being 4.7 years from LS diagnosis; 2 (4.3%) are alive with active LS and 37 (78.7%) died from LS, their original disease, or complications of treatment of LS (Fig 1B). For those alive in CR (n=8), 7 (87.5%) proceeded to a consolidative SCT for treatment of LS and 1 patient, an octogenarian remains in remission after AML directed therapy. Conclusions: LS is emerging as an important mechanism of immune escape following immunotherapy. Project EVOLVE provides the largest dataset of patients with LS to date. This systematic, international effort reveals substantial complexity and variability in diagnosing and managing LS. Unfortunately, outcomes following LS are grim, underscoring the critical need to identify risk factors of and optimal therapies for this leukemia transformation.

A Phase I Study of Venetoclax in Combination with Inotuzumab Ozogamicin for Relapsed or Refractory ALL in Adults

Introduction Adults with acute lymphoblastic leukemia (ALL), especially those who are older and/or have unfavorable disease biology frequently relapse. Inotuzumab ozogamicin (INO), an anti-CD22 monoclonal antibody covalently linked to calicheamicin, is approved for treatment of relapsed or refractory (R/R) CD22+ ALL. Although INO induces measurable residual disease negative (MRD-neg) complete remission (CR) in >75% of patients (pts), the duration of response (DOR) is limited (<6 months). Pre-clinical work demonstrates that malignant lymphoblasts are BCL2-dependent (Del Gaizo Moore Blood 2008). Initial results of the BCL2 inhibitor venetoclax (VEN) combination therapies for ALL are promising (Jain ASH 2019, Pullarkat Cancer Discov 2021). Due to distinct mechanisms of activity, non-overlapping toxicity, and possible synergy (Kirchhoff Blood 2021), we hypothesized that INO and VEN can be safely and effectively combined. Study Design and Eligibility This is an investigator-sponsored phase I study (NCT05016947) with a 3+3 dose escalation design. The primary objective is to determine the maximum tolerated dose (MTD) of VEN plus INO at the FDA-approved doses in adults with R/R CD22+ ALL. Secondary objectives assess efficacy (hematologic response, MRD by multi-parametric flow cytometry [MFC] threshold <0.01%, and DOR). A 14 pt expansion cohort is planned. Dose-limiting toxicity (DLT) is defined as treatment-related CTCAE v5 non-heme toxicity grade 3+, sinusoidal obstructive syndrome (SOS) any grade, or liver toxicity during Induction (Ind) Cycle (C) 1 and/or failure of hematologic recovery (ANC ≥500, Plts ≥25 K/mL) by day 42 of Ind C1 in responding pts. Eligibility: ≥18 years (yrs); ALL (≥5% blasts) or LBL (marrow involvement not required); CD22 expression on ≥20% of blasts; R/R to one or more cycles of CC or in the case of Philadelphia-chromosome (Ph)-positive ALL, failed by two or more TKIs, or refractory to/ineligible for ponatinib; ≥60 days from bone marrow transplant (BMT), and off immune suppression ≥2 weeks; adequate liver and kidney function; no history of liver disease including SOS; blast count ≤25 K/mL prior to treatment; no symptomatic central nervous system (CNS) disease. Pts with prior INO or VEN treatment for R/R ALL are excluded. Regimen Pts are treated with a VEN Ramp-Up over 3 days (DL1: 100/200/200; DL2: 100/200/400) followed by Induction C1: IV INO days 1, 8, 15 (0.8/0.5/0.5 mg/m2) and VEN daily per DL days 1-21. DEX 10 mg/m2 IV/PO is administered during Ramp-Up and Ind C1 days 1-4 (7 days total). Pts with >5% blasts after C1 receive Ind C2 without DEX. Pts with <5% blasts after Ind (1 or 2 cycles) continue with up to four 28-day consolidation cycles (INO 0.5 mg/m2 days 1, 8, 15 plus VEN per DL days 1-21). Pts receive CNS prophylaxis with intrathecal chemotherapy. Results The 2-level dose-escalation phase has been completed; we enrolled 9 pts (5 female/4 male) with Ph-negative (n=7) or Ph-positive (n=2) ALL. The median age was 45 yrs (range 25 -74). Pts had received 1 (n=5) or 2 (n=4) prior lines of therapy. DL1 enrolled 3 pts without DLT. All DL1 pts achieved an MRD-neg CR and bridged to consolidation: 1 pt XRT to involved disease site (ankle) after 3 cycles and 2 pts to BMT after 2 cycles (Table). Three pts enrolled at DL2; no DLTs were observed. All achieved an MRD-neg CR. Two pts bridged to consolidation (donor lymphocyte infusion after 3 cycles, blinatumomab after 5 cycles) while 1 pt with KMT2A-r ALL experienced early relapse with lineage switch during second cycle. A confirmatory cohort of 3 pts enrolled at DL2 without DLT. All achieved a CR (n=2, MRD-neg; n=1, MRD-pos). The pt with MRD-pos CR as best response proceeded to blinatumomab as bridge to BMT after 2 cycles with 2 pts continuing treatment (currently cycle 2). Overall, CR was achieved in 100% (9/9) of pts with 7/8 (87%) achieving MRD-negativity. With median follow-up of 195 days (95% CI 80 - NR), 2 pts have relapsed 48 (on treatment) and 177 (after BMT) days from registration while other pts remain alive in CR. Non-heme grade 3-4 AEs include febrile neutropenia (2), DIC (1), tumor lysis syndrome (1), anxiety (1), hypertension (1), and hypotension (1). There have been no SOS. There were no delays in count recovery during C1, with median time from C1 to C2 being 30 days (range 27-42). Conclusion VEN 400mg x 21 days in combination with INO at standard doses was well tolerated and declared the recommended phase 2 dose (RP2D) with a CR rate of 100%.

Retrospective Immunophenotypic Profile of Acute Leukemias in the State of Rio Grande Do Norte, Northeast, Brazil

INTRODUCTION:The main acute leukemias (ALs) are lymphoid (B or T lineage ALL) and myeloid (AML), in addition to biphenotype AL (BAL) and undifferentiated ALL (IAL), which are rarer. This variation reflects on therapy, requiring methods to characterize these leukemias. Thus, methods such as immunophenotyping by flow cytometry (ICF), multiparametric and quantitative, help in the follow-up of these neoplasms. Objective: The aim of this study was to demonstrate the immunophenotypic diagnostic profiles in patients with AL in Rio Grande do Norte, state, Brazil by FC. Methodology: Bone marrow and peripheral blood samples from 371 patients clinically diagnosed with AL were investigated by CF, using a panel of monoclonal antibodies for the detection of lymphoid (B, T and NK) and myeloid antigens, as well as markers related to cell immaturity such as CD34, Terminal deoxynucleotidyl Transferase (TdT).At the same time, information related to clinical data, age group and sex of patients was also collected. Results: Of all LA investigates 71 were B cell-ALL, 55 T cell-ALL, 239 AML, 04 BAL and 02 IAL. Regarding the immunophenotypic pattern, the main subgroups in relation to age group and were evidenced through the FC. 06/71 were Pro-B ALL, 48/71 Common ALL, 06/71 cyt mü chain ALL and 11/71 mature sIgM+ B-ALL. In B-cell lineage ALL, 51 (71.8%) of the cases were children (less than or equal to 18 years old) and 20 (28.2%) adults (over 18 years old). In T-cell ALL, 9 (16.4%) were pre-T ALL, 12 (21.8%) intermediate ALL and 29 (52.7%) medullary T-type ALL, corresponding to a total of 36.4% children and 63.6% adults. In AML, there was a predominance of involvement in adult patients, corresponding to47 (80.7%) of cases and 192 (80.3%) in children. Biphenotypic and undifferentiated AL were detected in adult patients. DISCUSSION: The B cell-ALL were characterized by natural arrest of B-cells precursors based on the expression of antigens related to this lineage in: i) Pro-B ALL (CD19+, HLA-Dr+, CD34+, TdT+); ii) Common ALL (CD10+, CD19+, cCD79a+, cytCD22+, HLA-Dr+, TdT+/- and CD34+/-); iii) cyt mü chain+ ALL (cyt IgM+, CD19+, HLA-Dr+, cytCD79a+, cytCD22+, CD10-, CD34-) and iv) mature B-ALL (sIgM+, sCD22+, CD10+/-, CD20+ and kappa+ or lambda+). The T-ALL were classified into: i) pre-T-ALL (CD7+, cytCD3+, HLADr+, CD34+ and TdT+), ii) intermediate T-ALL (CD1a+, CD2+, CD7+, cytCD3+, TdT+ and HLADr+, CD4+/CD8+) and Medullary T-ALL (CD4+ or CD8+, sCD3+, CD1a-, TdT+, HLADr+/- and CD34-). It was also possible to observe the aberrant expression of myeloid antigens in B and T-ALL, such as CD13, CD33 and CD66b, mainly related to ALL in the context of KMT2A rearrangement with lineage switching. In addition to transferrin receptors (CD71) and IL-2 receptor (CD25), which have been reported in the context of ALL BCR+/ABL+, as well as the presence of the Philadelphia chromosome. In AMLs, the most observed subtypes were those with little differentiation and with a monocyte component. (CD64+/CD14+). CD15+/CD34+ expression was also observed, indicative of asynchrony of maturation, and phenotypic aberrancy of lymphoid antigens in some cases, predominantly CD7. In addition to other antigens such as CD2 and CD19, CD19 and CD56, In addition, ALL was more frequent in children and males, while AML in adults and females in general, with a predominant incidence in males among adult patient. Conclusion: When properly applied, ICF can have a great impact on the diagnosis, classification and prognostic analysis of neoplastic processes.

Single Cell Transcriptomic Analysis of B-Cell Acute Lymphoblastic Leukemia (B-ALL) Reveals Correlation between Leukemia Cell State, Genetic Alterations and Clinical Outcome

I.I., A.G.X.Z, Q.G., L.G.P.: co-first authors; J.E.D., C.G.M.: co-senior authors Introduction: Genomic analyses of bulk ALL samples have improved our understanding of the genetic basis and risk stratification of B-ALL, but do not directly examine intratumor heterogeneity or enable inference of leukemia developmental state and cell of origin. Methods: We profiled 89 B-ALL samples by single-cell RNA-seq (scRNA-seq) (10X Genomics 5'v2) and compared them to a scRNA-seq reference map of normal human B-cell development. Results: Analysis of heterogeneity of inferred DNA copy number alterations at single cell level showed that aneuploid ALL with near haploidy and hyperdiploid harbor chromosomal losses or gains in all blasts, consistent with early, synchronous origin rather than sequential accumulation. A subset of TCF3::PBX1 samples harbored clones with progressive overexpression of genes surrounding PBX1 which scWGS confirmed caused by accumulation of DNA gains. Extensive intra-sample heterogeneity was observed by non-negative matrix factorization and driven by five consensus gene expression signatures: cell cycle (S; G2/M), metabolism, differentiation and inflammation. Signature composition refined leukemic subtyping; for example, within DUX4-r B-ALL two groups were discerned: one with higher inflammation score, expression of stem cell genes and worse outcome and one with lower inflammation score and expression of Pro-B related genes. To understand variation in developmental states among B-ALL blasts, we first developed an atlas of human B cell development. Marker genes elucidated from bulk RNA-seq on purified human HSC, MPP, LMPP, MLP, CLP, Pre-Pro-B, Pro-B, Pre-B, and B populations guided the development of a new scRNA-seq atlas of human B cell development comprising 130,085 cells spanning 90 fetal, pediatric, and adult samples from 8 studies (Fig 1A). Unexpectedly, we found that human CLPs retain transcriptional programs governed by CEBPA; in vitro and in vivo functional assays validated the capacity of CLPs for extensive myeloid differentiation prior to lineage restriction at the Pro-B stage. We next mapped our 89 B-ALL sc samples to precise cellular states along B cell development and clustered samples by leukemia cell composition. As expected, most samples exhibited similarity of GEP to that of pro-B cells, and this was most pronounced among hyperdiploid samples. High pre-B abundance was observed in MEF2D-r and TCF3::PBX1, while an HSC/MPP/LMPP enriched cluster encompassed ZNF384-r and a subset of DUX4-r B-ALL. Notably, one ZNF384-r B-ALL patient with LMPP involvement at diagnosis retained these LMPP-like cells at relapse despite a lineage switch to AML. Finally, we identified a group of patients with high early lymphoid (MLP, CLP, Pre-Pro-B) abundance representing a subset of Ph+ and KMT2A-r samples (Fig. 1B). We next utilized marker genes from each B-ALL cell state to estimate their relative abundance within a bulk RNA-seq cohort of 2046 B-ALL patients. In addition to validating these associations between genomic class and leukemia cell state, we identified age-dependent patterns of B-ALL state involvement wherein early lymphoid involvement is highest in infancy and adulthood while Pro-B involvement is highest in childhood. In childhood, high-risk disease is associated with higher early lymphoid ( P=4.9e-12) and lower Pro-B ( P=0.0085) abundance. Further, patients with residual disease (RD) levels above 1% are enriched for higher upfront early lymphoid abundance ( P=1e-7) and lower Pro-B ( P=4.1e-5) and Pre-B ( P=2.2e-11) abundance compared to patients with RD < 0.01%. Finally, B-ALL cell state improved resolution of previously reported transcriptional heterogeneity within defined genomic classes. Early lymphoid abundance discerns between two subtypes within Ph+ (Kim Nat Gen 2023) with AUC=0.953 ( P=2.4e-9), as well as two subtypes within KMT2A-r (Brady Nat Gen 2022) with AUC=0.996 ( P=1.1e-6). Notably we find that KMT2A::AFF1 fusions produce early lymphoid-enriched disease compared to KMT2A::MLLT3 ( P=0.0024) and KMT2A::MLLT10 ( P=0.0037), wherein the latter two fusions exhibit Pre-B enrichment ( P=0.025 and P=0.0037, respectively). Conclusions: Understanding variation in transcriptional programs and developmental states of B-ALL blasts by sc transcriptome refines existing clinical and genomic classifications and provides novel prognostic markers.

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (<40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived >60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

Lineage Switch Subsequently Developed after Isolated Extramedullary Relapse in Children with Acute Myeloid Leukemia

Lineage Switch Subsequently Developed after Isolated Extramedullary Relapse in Children with Acute Myeloid Leukemia

Mechanisms of resistance to CAR-T cell immunotherapy: Insights from a mathematical model

Chimeric Antigen Receptor (CAR)-T cell therapy long-term follow-up studies revealed non-durable remissions in a significant number of patients. Some of the mechanisms underlying these relapses include poor CAR T cell cytotoxicity or persistence, as well as antigen loss or lineage switching in tumor cells. In order to investigate how antigen-mediated resistance mechanisms affect therapy outcomes, we develop a mathematical model based on a set of integral-partial differential equations. Using a continuous variable to describe the level of antigen expression of tumor cells, we recapitulated important cellular mechanisms across patients with different therapeutic responses. Fitted with clinical data, the model successfully captured the dynamics of tumor and CAR-T cells for several hematological cancers. Furthermore, the role played by these mechanisms are explored with regard to different biological scenarios, such as pre-existing or aquired mutations, providing a deeper understanding of key factors underlying resistance to CAR-T cell immunotherapy.

Abstract P3159: Exosomes Derived From Podoplanin Positive Cells Induce Endo-Mt And Myofibroblast Transformation In Healthy Mouse Heart And After Ischemic Injury

Specific understanding of the differences in cell communications between physiologicaland pathological conditions harbor the understanding of mechanisms to recover theequilibrium in the injured tissue and restore the organ function. Neurohumoral factorsreleased after myocardial infarction (MI) induce the expression of platelet aggregation-inducing type I transmembrane glycoprotein named Podoplanin (PDPN) in mesenchymalstromal cells, which pivot their structural support function and actively participate in theremodeling of the ischemic tissue releasing an abundance of extracellular vesicles calledexosomes. Exosomes (exo) derived from activated PDPN+ cells conditioned media wereused in vivo for the treatment of healthy mouse hearts, and in vitro for the treatment ofmouse cardiac endothelial cells (mCECs), NHI-3T3 and adult cardiac fibroblast. Ourongoing experiments showed that in vivo, PDPN-exo could independently alter healthyheart physiology and structure; mice hearts treated with PDPN-exo developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increaseof the end diastolic and systolic volumes. The fibrotic area was characterized by ECMdeposition, increased lymphatic vessels and infiltrating CD45+ cells. In vitro, PDPN-exoreprogramed mCECs, NHI-3T3 and adult cardiac fibroblast, upregulating the expressionof fibrotic markers suggesting an Endothelial-Mesenchymal Transition (EndoMT) andmyofibroblast transformation. Proteomic analysis of PDPN-exo suggests these transitionsmay depend upon the delivery of NOTCH1 via exosomes and the cleavage of it throughγ-Secretase. Experiments of gain and loss of NOTCH1 function showed thatoverexpression of NOTCH1 intracellular domain (NICD1) in vitro was sufficient to derivea lineage switching in mCECs and fibroblasts on the contrary in vivo delivery and in vitrotreatment with exosomes derived from PDPN+ cells silenced for NOTCH1 or treatmentwith γ-Secretase inhibitor did not alter the heart structure and function or the endothelialcells and fibroblast lineage. Thus, NOTCH1 delivery via PDPN-exo after MI play a majorrole in scar remodeling and its biology can be pharmacologically modulated.

Myeloid lineage switch following CD7-targeted chimeric antigen receptor T-cell therapy in relapsed/refractory T-cell acute lymphoblastic leukemia.

Not available.

Clonal origin of KMT2A wild-type lineage-switch leukemia following CAR-T cell and blinatumomab therapy

Children with acute lymphoblastic leukemia (ALL) undergoing anti-CD19 therapy occasionally develop acute myeloid leukemia (AML). The clonal origin of such lineage-switch leukemias1–4 remains unresolved. Here, we reconstructed the phylogeny of multiple leukemias in a girl who, following multiply relapsed ALL, received anti-CD19 cellular and antibody treatment and subsequently developed AML. Whole genome sequencing unambiguously revealed the AML derived from the initial ALL, with distinct driver mutations that were detectable before emergence. Extensive prior diversification and subsequent clonal selection underpins this fatal lineage switch. Genomic monitoring of primary leukemias and recurrences may predict therapy resistance, especially regarding anti-CD19 treatment.

The SWI/SNF complex member SMARCB1 supports lineage fidelity in kidney cancer

Lineage switching can induce therapy resistance in cancer. Yet, how lineage fidelity is maintained and how it can be lost remain poorly understood. Here, we have used CRISPR-Cas9-based genetic screening to demonstrate that loss of SMARCB1, a member of the SWI/SNF chromatin remodeling complex, can confer an advantage to clear cell renal cell carcinoma (ccRCC) cells upon inhibition of the renal lineage factor PAX8. Lineage factor inhibition-resistant ccRCC cells formed tumors with morphological features, but not molecular markers, of neuroendocrine differentiation. SMARCB1 inactivation led to large-scale loss of kidney-specific epigenetic programs and restoration of proliferative capacity through the adoption of new dependencies on factors that represent rare essential genes across different cancers. We further developed an analytical approach to systematically characterize lineage fidelity using large-scale CRISPR-Cas9 data. An understanding of the rules that govern lineage switching could aid the development of more durable lineage factor-targeted and other cancer therapies.

Chimeric Antigen Receptor T Cells as Salvage Therapy for Post-Chimeric Antigen Receptor T Cell Failure

Outcomes for post-chimeric antigen receptor (CAR) T cell therapy (CART) relapse are poor. The utilization of a unique CAR T cell construct for post-CART failure is increasing, but this approach is not well described. In this study, with CART-A the first unique CAR T cell construct received and CART-B the second, the primary objective was to characterize outcomes following CART-B. Secondary objectives included evaluating safety and toxicity with sequential CART infusions; investigating the impact of potential factors, such as antigen modulation and interval therapy, on CART-B response; and characterizing long-term outcomes in patients receiving multiple CARTs. This was a retrospective review (NCT03827343) of children and young adults with B cell acute lymphoblastic leukemia (B-ALL) undergoing CART therapy who received at least 2 unique CART constructs, excluding interim CART reinfusions of the same product. Of 135 patients, 61 (45.1%) received 2 unique CART constructs, including 13 who received >2 CARTs over time. Patients included in this analysis received 14 distinct CARTs targeting CD19 and/or CD22. The median age at CART-A was 12.6 years (range, 3.3 to 30.4 years). The median time from CART-A to CART-B was 302 days (range, 53 to 1183 days). CART-B targeted a different antigen than CART-A in 48 patients (78.7%), owing primarily to loss of CART-A antigen target. The rate of complete remission (CR) was lower with CART-B (65.5%; 40 of 61) than with CART-A (88.5%; 54 of 61; P=.0043); 35 of 40 (87.5%) CART-B responders had CART-B targeting a different antigen than CART-A. Among the 21 patients with a partial response or nonresponse to CART-B, 8 (38.1%) received CART-B with the same antigen target as CART-A. Of 40 patients with CART-B complete response (CR), 29 (72.5%) relapsed. For the 21 patients with evaluable data, the relapse immunophenotype was antigennegative in 3 (14.3%), antigendim in 7 (33.3%), antigenpositive in 10 (47.6%), and lineage switch in 1 (4.8%). The median relapse-free survival following CART-B CR was 9.4 months (95% confidence interval [CI], 6.1 to 13.2 months), and overall survival was 15.0 months (95% CI, 13.0 to 22.7 months). Given the limited salvage options for post-CART relapse, identifying optimizing strategies for CART-B is critical. We raise awareness about the emerging use of CART for post-CART failure and highlight clinical implications accompanying this paradigm shift.

Plasticity of lineage switch in B-ALL allows for successful rechallenge with CD19-directed immunotherapy.

Plasticity of lineage switch in B-ALL allows for successful rechallenge with CD19-directed immunotherapy.

Abstract B025: MicroRNA regulators of neuroendocrine differentiation of prostate cancer

Abstract Background: Therapy-induced neuroendocrine prostate cancer (NEPC) is an extremely aggressive variant of castration-resistant prostate cancer (CRPC) that is increasing in incidence with the widespread use of second generation of androgen receptor (AR)-pathway inhibitors (APIs) such as Enzalutamide (ENZ) and Abiraterone. This aggressive variant arises from CRPC-Adenocarcinomas (CRPC-Adeno) via a reversible trans-differentiation process, referred to as neuroendocrine differentiation (NED) wherein cells undergo a lineage switch and exhibit neuroendocrine (NE) features, characterized by expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA) and synaptophysin (SYP). There is an urgent need of identifying novel molecular markers to assess emergence of NED in CRPC patients. We demonstrated that progression of advanced CRPC with adenocarcinoma characteristics (CRPC-Adeno) to therapy-induced, androgen-independent NE (CRPC-NE) states is associated with a characteristic set of miRNA alterations that promote plasticity of advanced prostate adenocarcinomas to NEPC (Bhagirath et al., Oncogene, 2020). Importantly, we could develop a ‘novel miRNA classifier’ to robustly stratify CRPC-NE tumors from CRPC-Adenocarcinomas. Here we further validate the classifier in independent clinical cohorts and deduce the optimal miRNA genes required for NEPC diagnosis. We further study the functional significance of miR-28-3p, a miRNA identified by sequencing, in NEPC. Methods: Human clinical samples with corresponding clinical information were procured from two independent sites of Prostate Cancer Biorepository Network (PCBN). Samples included human CRPC-adeno vs CRPC-NE. FFPE sections from these clinical samples were microdissected, RNA were extracted and small RNA sequencing was performed using an Illumina NextSeq 500 platform. Sequencing data were analyzed and machine learning algorithms were applied (random forest machine learning technique with leave-pair-out cross validation (LPOCV)). The performance of classifier was measured using receiver operating characteristic (ROC) analysis with area under the curve (AUC) as the primary evaluation metric. The functional significance of miR-28-3p was studied by its overexpression and knockdown in PCa cell lines followed by functional assays. Results: Unsupervised analysis of sequencing data by principal component analyses (PCA) revealed distinct clustering of the CRPC-NE tumors from CRPC-Adenocarcinomas based on miRNA profiles suggesting that miRNA profiles can be used to stratify these tumor types. Our analyses showed that a set of 5 miRNAs of the classifier are important in distinguishing between CRPC-Adeno vs CRPC-NE with an AUC=0.8318. Further, miR 28-3p potentially has a biphasic role in PCa, where it is oncogenic in early stages and acts as a tumor suppressor in late stage PCa. Conclusions: A ‘5- miRNA’ classifier was validated to be of significance in two independent validation cohorts. We propose this miRNA classifier as an important tool for diagnosing NED in CRPC patients. Citation Format: Amritha Sreekumar, Sharanjot Saini. MicroRNA regulators of neuroendocrine differentiation of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B025.

Comprehension characterization of prostate cancer cell lines identified JAK-STAT3 signaling in lineage-switched non-neuroendocrine cells.

e17005 Background: Prostate cancer (PCa) is the most diagnosed cancer type among American men. Androgen receptor (AR) targeted therapy as the gold standard of PCa has been challenged by treatment resistance. Lineage plasticity enables PCa cells to transit from prostate luminal epithelia to alternative lineages to bypass their dependency on AR signaling. Previously we established prostatic HOX code to study the prostatic lineage. We found that cells with neuroendocrine (NE) phenotype have a lineage switch. Besides, a subgroup of AR negative, NE negative samples also display lineage switch, among which is DU145, a human PCa cell line. Methods: RNA-seq data of PCa cell lines were obtained from GEO then aligned and quantified using human GRCh38 genome with STAR-Salmon pipeline. Downstream differential expression and enrichment analysis was performed with R. Immunohistochemistry (IHC) was used to assess protein levels on cell line derived xenografts. Results: 45 RNA-seq datasets of 6 prostate adenocarcinoma cell lines (VCaP, LNCaP, C4-2, 22RV1, PC3 and DU145) and 8 RNA-seq datasets of neuroendocrine prostate cancer cell line (H660) passed quality control. Principle component analysis indicates that samples from the same cell line had similar transcriptomes. Differential expression analysis followed by gene function enrichments analysis identified the marker genes and key signaling pathways of each cell line. HOX code analysis confirmed previous conclusion that all PCa cell lines but DU145 and H660 samples maintained prostatic HOX code. IHC staining on cell line-derived xenografts confirmed the protein expression of selected marker genes (TUBB2B for H660, HGF for VCaP and 22RV1, CXCL1 and KRT7 for DU145 and PC3). The three AR-negative PCa cell lines were further characterized in terms of AR/ NE activity scores, transcriptome identities, HOX codes. These analyses demonstrated that PC3 is a prostate progenitor like cell line, H660 is a lineage-switched NE cell line, and DU145 is a lineage-switched non-NE cell line. Further, signaling enrichment analysis revealed that DU145 cells had higher JAK-STAT3 signaling activity compared to other cell lines. IHC staining detected strong p-STAT3 signals on DU145-derived tumors but not on any other cell line-derived tumors. Conclusions: Human PCa cell lines were fully characterized based on their transcriptomes with high sample numbers. PC3 represents cells dedifferentiated into progenitor stage. DU145 and H660 represent cells that further trans-differentiate into alternative lineages. Neuron master regulators drive H660 cells into NE lineage and STAT3 drives DU145 cells into a non-NE lineage. DU145, representing a non-NE PCa class that have lost prostatic lineage, would be a suitable model system to study JAK-STAT targeting therapy.

Lineage switch of B-acute lymphoblastic leukemia with t(11;19)(q23;p13.3) into acute myeloid leukemia with monocytic differentiation after induction chemotherapy.

Lineage switch of B-acute lymphoblastic leukemia with t(11;19)(q23;p13.3) into acute myeloid leukemia with monocytic differentiation after induction chemotherapy.

Repurposing ketotifen as a therapeutic strategy for neuroendocrine prostate cancer by targeting the IL-6/STAT3 pathway.

Neuroendocrine prostate cancer (NEPC), a highly aggressive subtype of prostate cancer displaying resistance to hormone therapy, presents a poor prognosis and limited therapeutic options. Here, we aimed to find novel medication therapies for NEPC and explore the underlying mechanism. A high-throughput drug screening utilizing an FDA-approved drug library was performed and ketotifen, an antihistamine agent, was identified as a potential therapeutic candidate for NEPC. The whole-transcriptome sequencing analysis was conducted to explore mechanism of ketotifen inhibitory in NEPC. Multiple cell biology and biochemistry experiments were performed to confirm the inhibitory effect of ketotifen in vitro. A spontaneous NEPC mice model (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f)was used to reveal the inhibitory effect of ketotifen in vivo. Our in vitro experiments demonstrated that ketotifen effectively suppressed neuroendocrine differentiation, reduced cell viability, and reversed the lineage switch via targeting the IL-6/STAT3 pathway. Our in vivo results showed that ketotifen significantly prolonged overall survival and reduced the risk of distant metastases in NEPC mice model. Our findings repurpose ketotifen for antitumor applications and endorse its clinical development for NEPC therapy, offering a novel and promising therapeutic strategy for this formidable cancer subtype.

Phenotypic and genotypic infidelity in B-lineage neoplasms, including transdifferentiation following targeted therapy: Report from the 2021 SH/EAHP Workshop.

Session 2 of the 2021 Society for Hematopathology and European Association for Haematopathology Workshop collected examples of lineage infidelity and transdifferentiation in B-lineage neoplasms, including after targeted therapy. Twenty cases were submitted. Whole-exome sequencing and genome-wide RNA expression analysis were available on a limited subsample. A diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) was rendered on at least 1 biopsy from 13 patients. There was 1 case of acute myeloid leukemia (AML); the remaining 6 cases were mature B-cell neoplasms. Targeted therapy was administered in 7 cases of B-ALL and 4 cases of mature B-cell neoplasms. Six cases of B-ALL underwent lineage switch to AML or mixed-phenotype acute leukemia at relapse, 5 of which had rearranged KMT2A. Changes in maturational state without lineage switch were observed in 2 cases. Examples of de novo aberrant T-cell antigen expression (n = 2) were seen among the mature B-cell lymphoma cohort, and their presence correlated with alterations in tumor cell gene expression patterns. This cohort of cases enabled us to illustrate, discuss, and review current concepts of lineage switch and aberrant antigen expression in a variety of B-cell neoplasms and draw attention to the role targeted therapies may have in predisposing neoplasms to transdifferentiation as well as other, less expected changes in maturational status.